Dr Nicoletta Galeotti
email: nicoletta.galeotti@unifi.it

Elisa Vivoli

Anna Rita Bilia

Francesco Franco

Vincieri

Carla Ghelardini

St John's Wort reduces neuropathic pain through a hypericin-mediated inhibition of the protein kinase C γ and ε activity

Keywords: hypericum perforatum, St. John's Wort, neuropathic pain, hypericin, hyperforin, PKC

Current pharmacological treatments for neuropathic pain have limited efficacy and severe sideeffect limitations. St. John's Wort (SJW) is a medicinal plant, mainly used as antidepressant, with a favourable side-effect profile. We here demonstrate the ability of SJW to relieve neuropathic pain in rat models. The antihyperalgesic profile and mechanism of action of SJW and its main components was studied in two rat models of neuropathic pain: the chronic constriction injury and the repeated administration of oxaliplatin. SJW, acutely administered at low doses (30-60 mg kg -1 p.o.), reversed mechanical hyperalgesia with a prolonged effect, being effective up to 180 min after injection.

Further examinations of the SJW main components revealed that hyperforin and hypericin were responsible for the antihyperalgesic properties whereas flavonoids were ineffective. The effect of SJW on the PKC expression and activation was investigated in the periaqueductal grey (PAG) area by immunoblotting experiments. Mechanistic studies showed a robust over-expression and hyperphosphorylation of the PKC-γ (227.0±15.0 % of control) and -ε (213.9±17.0) isoforms in the rat PAG area. A single oral administration of SJW produced a significant decrease of the PKC-γ (131.8±10.0) and -ε (105.2±12.0) phosphorylation in the PAG area due to the presence of hypericin.

Furthermore, SJW showed a dual mechanism of action since hyperforin antinociception involves an opioid-dependent pathway. Rats undergoing treatment with SJW and purified components did not show any behavioural side effects or signs of altered locomotor activity. Our results indicate SJW as a prolonged antihyperalgesic treatment through inhibition of PKC isoforms and their phosphorylation.

A c c e p t e d M a n u s c r i p t 2 Abbreviations: CCI, chronic constriction injury; CHL, chloroformic fraction; i.c.v., intracerebroventricular; i.p., intraperitoneal; MET, methanolic fraction; OXA, oxaliplatin repeated administration; p.o.: per os; PAG, periaqueductal grey area; PKC, protein kinase C; PMA, phorbol-12-myristate-13-acetate; SJW, St. John's Wort; s.c., subcutaneous A c c e p t e d M a n u s c r i p t

Introduction

Neuropathic pain can arise from a wide variety of injuries to peripheral or central nerves, including metabolic disorders, traumatic injury, inflammation, and neurotoxicity, and is characterized by spontaneous pain, hyperalgesia and allodynia which can persist long after the initial injury is resolved [START_REF] Woolf | Neuropathic pain: aetiology, symptoms, mechanisms, and management[END_REF]. Common causes of neuropathy are diabetes, herpes zoster infections, chronic or acute trauma, and neurotoxins. Neuropathic pain occurs frequently in cancer as it may result from tumor invasion of nervous tissue, surgical nerve damage during tumor removal, radiation-induced nerve damage or as a side effect of many chemoterapeutic drugs [START_REF] Cruccu | EFNS guidelines on neuropathic pain assessment[END_REF][START_REF] Hansson | Pharmacological treatments of peripheral neuropathic pain conditions based on shared commonalities despite multiple etiologies[END_REF] .

The underlying molecular mechanisms of neuropathic pain are still not completely understood, and as a consequence, treatment is unsatisfactory in many cases [START_REF] Hansson | Pharmacological treatments of peripheral neuropathic pain conditions based on shared commonalities despite multiple etiologies[END_REF][START_REF] Sindrup | Efficacy of pharmacological treatments of neuropathic pain: an update and effect related to mechanism of drug action[END_REF]. Despite the large number of approved analgesic drugs, effective treatment of chronic pain is still often unattainable due to the unsatisfactory performance of the available drugs and due to the negative side effects from the drugs [START_REF] Kingery | A critical review of controlled clinical trials for peripheral neuropathic pain and complex regional pain syndromes[END_REF][START_REF] Koltzenburg | Painful neuropathies[END_REF]. Neuropathic pain is generally insensitive to non-steroidal anti-inflammatory drugs and relatively resistant to opioids, but can be treated by high opioid doses [START_REF] Kalso | Opioids in chronic non-cancer pain: a systematic review of efficacy and safety[END_REF] with a high incidence of untoward side effects. The suggested treatment strategies for neuropathic pain management are represented by tricyclic antidepressants (TCAs) [START_REF] Wallace | Diagnosis and treatment of neuropathic pain[END_REF], that represent the first medication category proved to be effective for neuropathic pain in clinical trials [START_REF] Davis | Peripheral diabetic neuropathy treated with amitriptyline and fluphenazine[END_REF] and still are a first choice treatment, anticonvulsants [START_REF] Dworkin | Advances in neuropathic pain: diagnosis, mechanisms and treatment recommendations[END_REF][START_REF] Owen | Pregabalin: its efficacy, safety and tolerability profile in fibromyalgia syndrome[END_REF], antiarrythmics [START_REF] Rowbotham | Lidocaine patch: double-blind controlled study of a new treatment method for post-herpetic neuralgia[END_REF]. All these drugs have limited efficacy and have severe side effects. In particular, TCAs induce numerous untoward effects such as sedation, anticholinergic effects like dry mouth, constipation and postural hypotension, weight gain, greater risk to develop myocardial infarction [START_REF] Beniczky | Evidence-based pharmacological treatment of neuropathic pain syndromes[END_REF][START_REF] Cohen | Excess risk of myocardial infarction in patients treated with antidepressant medications: association with use of tricyclic agents[END_REF]. Therefore, for an effective treatment of neuropathic pain, there is still a need to obtain therapeutics which possess a greater level of tolerability and safety.

Hypericum perforatum L., commonly known as St. John's Wort (SJW), has been proven to relieve mild-to-moderate forms of depression and tests have also shown SJW to have favourable side effects [START_REF] Linde | St John's wort for depression[END_REF]. The most common SJW preparations used are hydroalcoholic extracts of the aerial portion of the plant that contain at least ten different kinds of biochemical compounds [START_REF] Greeson | St. John's wort (Hypericum perforatum): a review of the current pharmacological, toxicological, and clinical literature[END_REF]. SJW A c c e p t e d M a n u s c r i p t 5 interacts with the monoaminergic system through different mechanisms: the MAO-inhibitory properties of SJW were mainly due to hypericin [START_REF] Suzuki | Inhibition of monoamine oxidase by hypericin[END_REF][START_REF] Kubin | Hypericin -The facts about a controversial agent[END_REF] whereas the inhibition of serotonin, dopamine and noradrenaline synaptosomal uptake is related to the presence of hyperforin [START_REF] Chatterjee | Hyperforin as a possible antidepressant component of hypericum extracts[END_REF][START_REF] Chatterjee | Antidepressant activity of hypericum perforatum and hyperforin: the neglected possibility[END_REF][START_REF] Muller | Hyperforin represents the neurotransmitter reuptake inhibiting constituent of hypericum extract[END_REF][START_REF] Gobbi | Hypericum perforatum L. extract does not inhibit 5-HT transporter in the rat brain[END_REF]. It has therefore been suggested that SJW may induce antidepressant activity through a mechanism similar to TCAs [START_REF] Nathan | The experimental and clinical pharmacology of St John's Wort (Hypericum perforatum L.)[END_REF]. This evidence may suggest that similar to TCAs, SJW can also relieve neuropathic pain. The aim of the present study was to investigate the antihyperalgesic properties of SJW in different animal models, which were suffering of neuropathic pain that was induced by chronic constriction injury of the sciatic nerve, or by the repeated administration of the chemotherapeutic agent oxaliplatin. The role of the SJW main constituents, hypericin, hyperforin and flavonoids, and their cellular and molecular effects in the modulation of the pain threshold was also investigated in comparison with the effects produced by the SJW dried extract in order to better elucidate the mechanism of action in this herbal plant.

A c c e p t e d M a n u s c r i p t 6

Materials and Methods

Animals

Male Sprague-Dawley albino rats (180-200 g) from Harlan (S.Piero al Natisone, Italy) were used.

Four rats were housed per cage. The cages were placed in the experimental room 24 h before the test for acclimatization. The animals were fed a standard laboratory diet and tap water ad libitum and kept at 23±1°C. The rats had a 12-h light/dark cycle, light at 7 AM. All experiments were carried out in accordance with the NIH Guide for the Care and Use of Laboratory animals. All efforts were made to minimize animal suffering, and to reduce the number of animals used.

Production of neuropathy

Chronic constriction injury:

A peripheral mono neuropathy was produced in adult rats by placing loosely constrictive ligatures around the common sciatic nerve according to the method described by Bennett and Xie [START_REF] Bennett | peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man[END_REF]. Rats were anesthetized with chloral hydrate. The common sciatic nerve was exposed at the level of the middle of the thigh by blunt dissection through biceps femoris. Proximal to sciatica's trifurcation, about 1 cm of the nerve was freed of adhering tissue and four ligatures (3/0 chromic tread) were tied loosely around it with about 1mm spacing. The length of the affected nerve was 4-5 mm long. Great care was taken to tie the ligatures such that the diameter of the nerve was barely constricted when viewed with 40x magnification. In shamoperated animals, an identical dissection was performed, but the sciatic nerve was not ligated. The ligation was made on the right paws and left paws were untouched. Experiments were carried out 14 days after surgery.

Oxaliplatin repeated treatment:

Oxaliplatin was dissolved in a 5% glucose solution at a concentration of 2.4 mg kg -1 . Oxaliplatin was administered i.p. for five consecutive days per week for three consecutive weeks according to a previously established protocol [START_REF] Cavaletti | Effects of different schedules of oxaliplatin treatment on the peripheral nervous system of the rat[END_REF]. The control group

Paw-pressure test

The instrument has a cone-shaped pusher and exerts a force which is applied at a constant rate (32 g per second) on the upper surface of the rat hind paw. The force is continuously monitored by a pointer which moves along a linear scale. The pain threshold is given by the force which first induces struggling from the rat. An arbitrary cut off value of 250 g was adopted. Those rats scoring less than 40 g or over 75 g in the pretest, performed before chronic constriction injury or oxaliplatin administration, were rejected (25%). The pain threshold of neuropathic animals was measured previously (pretest) and then 30, 60, 90, 120 and 180 min after administration of SJW and components.

Rota rod test

The apparatus consisted of a base platform and a rotating rod with a diameter of 3 cm and a nonslippery surface. The rod, 30 cm in length, was placed at a height of 15 cm from the base. Up to five rats were tested simultaneously on the apparatus, with a rod-rotating speed of 16 rpm. The integrity of motor coordination was assessed on the basis of the number of falls from the rod in 30 s. Those rats scoring less than three and more than six falls in the pretest were rejected (20%). Their performance time was measured previously (pretest) and then 60, 90 and 120 min after SJW, CHL and MET fractions or purified components administration. A total of 4-5 rats per group were tested.

I.c.v. injection technique

Intracerebroventricular (i.c.v.) administration was performed under ether anesthesia with isotonic saline as a solvent which has been previously described [START_REF] Galeotti | The phospholipase C-IP 3 pathway is involved in muscarinic antinociception[END_REF].

A c c e p t e d M a n u s c r i p t 8

Preparation of membrane and cytosol fractions

Rat periaqueductal grey (PAG) area was dissected and homogenized in a homogenizing buffer. The homogenate was centrifuged at 9,000 x g for 15 min at 4°C and the low speed pellet was discarded.

The supernatant (total proteins) was centrifugated at 100,000 x g for 60 min at 4°C. The resulting supernatant was the cytosol fraction, and the pellet was resuspended in the homogenizing buffer containing 0.2% (wt/vol) Triton X-100. The homogenate was kept at 4°C for 60 min with occasional stirring and then centrifugated at 100,000 x g for 60 min at 4°C. The resulting supernatant was used as the membrane fraction. Protein concentration of the cytosol and membrane fractions was quantified using Bradford's method (protein assay kit, Bio Rad Laboratories, Milan, Italy).

Immunoblot analysis

Membrane homogenates (10-50 µg) of naïve, untreated CCI and SWJ treated CCI rats were separated on 10% SDS-PAGE and transferred onto nitrocellulose membranes (100 min at 100 V) using standard procedures. The membranes were blocked in PBST (PBS containing 0.1% Tween) containing 5% nonfat dry milk for 90 min. Following washings, blots were incubated overnight at 4° C with specific antibodies (Santa Cruz Biotechnology, CA, USA) against PKCγ, pPKCγ, PKCε, pPKCε or ß-actin (1:1000 dilution). After being washed with PBST, the nitrocellulose membranes were incubated with goat anti-rabbit horseradish peroxidase-conjugated secondary antisera (1:5,000) and left for 90 min at room temperature. Blots were then extensively washed according to the manufacturer's instruction and developed using enhanced chemiluminescence detection system (Pierce, Milan, Italy). The exposition and developing times used were standardized for all the blots. Densitometric analysis of scanned images was performed on a Macintosh iMac computer using the public domain NIH Image program. Measurements in control samples were assigned a relative A c c e p t e d M a n u s c r i p t 9 value of 100%. Measurements were normalised relative to ß-actin, used as loading control.

HPLC-DAD and HPLC-MS drug analyses

To evaluate the content of SJW main components, a HPLC analysis was performed using a method described in the [START_REF] Brolis | Identification by highperformance liquid chromatography-diode array detection-mass spectrometry and quantification by high performance liquid chromatography-UV absorbance detection of active constituents of Hypericum perforatum[END_REF], modified for our experimental requirements. The identification of the constituents was performed using combined HPLC-diode array detection (DAD) analysis and HPLC-thermos pray mass s pectrometry. The mobile phase was a five-step linear solvent gradient CH3CN/CH3OH/H2O (pH 3.2, HCOOH) over a 60 min period at a flow rate of 1 ml/min. The quantification of the cons tituents was performed using rutin as an external s tandard and consideration of each cons tituent and the relative res ponse factor (RRF) with res pect to the rutin, as previously reported [START_REF] Brolis | Identification by highperformance liquid chromatography-diode array detection-mass spectrometry and quantification by high performance liquid chromatography-UV absorbance detection of active constituents of Hypericum perforatum[END_REF].

All the samples were analyzed in triplicate and a calibration graph with six data points of external standard was used.

Preparation of chloroform and methanol fractions

A solution of dried SJW extract (1 g) in 10 ml of chloroform was stirred for 3 hours in the dark. The mixture was filtered and the solution was evaporated to obtain a residue of 200 mg, containing the pholoroglucinols as confirmed by HPLC-DAD-ESI-MS analysis.

The residue present on the filter was washed with 10 ml of methanol. The methanol solution was evaporated and a residue of 700 mg was obtained. The HPLC-DAD-ESI-MS analysis showed in this fraction only the presence of flavonoids and naphtodianthrones. The following drugs were used: oxaliplatin (Sequoia Research Product Ltd, USA), naloxone hydrochloride, hypericin, hyperforin, hyperoside, quercetin, amentoflavone, amitriptyline hydrochloride (Sigma, Milan, Italy); phorbol-12-myristate-13-acetate (PMA), (Calbiochem, Milan Italy); and carboxymethylcellulose sodium salt, (CMC) (Fluka Chemie GmbH, Steinheim, Germany). The other commercially available chemicals used were of the highest quality. SJW dried extract, chloroform fraction (CHL), methanol fraction (MET), hypericin, hyperforin, quercetin, hyperoside and amentoflavone were dissolved in a 1% CMC solution immediately before use and administered by oral gavage. Naloxone and amitriptyline were dissolved in isotonic (NaCl 0.9 %) saline solution, PMA was dissolved in 0.1% DMSO immediately before use. Drug concentrations were prepared to ensure that the necessary dose could be administered in a volume of 10 ml kg -1 by intraperitoneal (i.p.), per os (p.o.) injection or in a volume of 5 µl by i.c.v. injection.

Drugs

Drug administration

SJW dried extract, CHL and MET fractions, hypericin, hyperforin, quercetin, hyperoside and amentoflavone were administered 90 min before the Paw Pressure test, in correspondence to their maximum effect as determined by time-course experiments.

The SJW components were orally administered 90 min before the test at the following doses: hypericin 0.06 mg kg -1 , hyperforin 0.978 mg kg -1 , quercetin 0.249 mg kg -1 , hyperoside 1.905 mg kg - 1 amentoflavone 0.016 mg kg -1 . These doses correspond to the amount of each component present in a 30 mg kg -1 preparation of SJW dried extract. CHL and MET fractions were administered in a concentration containing the above-mentioned content of their main components, hyperforin and hypericin respectively. The effect of amitriptyline was evaluated 30 min after admistration. The doses and administration schedules of SJW and all other drugs used were chosen on the bases of time-course and dose-response experiments previously performed in our laboratory.

Statistical analysis

All experimental results are given as the mean ± S.E.M. An analysis of variance ANOVA, followed by Fisher's Protected Least Significant Difference procedure for post-hoc comparison, were used to verify significance between two means.

A c c e p t e d M a n u s c r i p t 12 3. Results

Composition of SJW dried extract, chloroform and methanol fractions.

The main components of SJW dried extract were flavonoids (12.72%), phloroglucinols (4.23%) and naphtodianthrones (0.32%), as reported in Table 1. In order to investigate the role of these components in the mechanism of action of SJW, a chloroform and a methanol fraction were obtained from the dried extract. The chloroform fraction (CHL) contained phloroglucinols whereas the methanol fraction (MET) showed the presence of naphtodiantrones and flavonoids representing a hypericin and flavonoid rich fraction (Table 1).

Antihyperalgesic/antinociceptive activity of SJW dried extract in rat models of neuropathic pain.

Two different rat models of neuropathic pain were used: chronic constriction injury of the sciatic nerve (CCI) and oxaliplatin repeated administration (OXA). CCI, 14 days after surgery, induced mechanical hyperalgesia (rat paw pressure test) as demonstrated by the reduced pain threshold in the hind paw affected by CCI in comparison with the controlateral uninjured leg (Figure 1a F [START_REF] Woolf | Neuropathic pain: aetiology, symptoms, mechanisms, and management[END_REF][START_REF] Chatterjee | Hyperforin as a possible antidepressant component of hypericum extracts[END_REF]44.631 p<0.001). Pain threshold values comparable to those recorded in the controlateral uninjured leg were detected in the sham-operated before (65.0±5.5) and after (64.6±3.3) a single oral administration of CMC. Same results were obtained in naïve rats (Figure 1b). Similarly to CCI, oxaliplatin repeated administration induced the development of neuropathic pain as illustrated by nociceptive behaviour as compared with the saline-treated control group. A statistically significant hyperalgesia appeared on day 21, persisted unmodified up to day 27 and then it diminished (Figure 1c).

A single administration of SJW dried extract dose-dependently (1-100 mg kg -1 p.o.) reversed the mechanical hyperalgesia in both models of neuropathic pain: CCI (Figure 1a;) and OXA (Figure ). The antihyperalgesic effect appeared at antinociceptive doses: the dose of 1 mg kg -1 p.o. was devoid of any effect, at 10 mg kg -1 p.o. SJW significantly increased the rat pain threshold peaking at 30 mg kg -1 p.o. At higher concentrations, the antihyperalgesic effect diminished showing a bellshaped trend (Figure 1a,d). The SJW antihyperalgesic effect was of similar intensity to that produced by amitriptyline (10 mg kg -1 i.p.; 80.3±4.2), used as reference drug. Time-course experiments showed that the antinociceptive (Figure 2a,c) and the antihyperalgesic (Figure 2b,c) effects of SJW (30 mg kg -1 p.o.) share the same profile: they both became statistically significant 30 min after oral administration, peaked at 90 min, persisted up to 120 min and disappeared at 180 min in all of the rat models tested for neuropathic pain. In the sham-operated rats SJW (30 mg kg -1 p.o.) increased the pain threshold (controlateral 110.1±3.1: sham-operated 108.8±5.6) with similar intensity to that observed in naïve rats (Figure 1b) and in the controlateral uninjured leg of CCI rats (Figure 2a).

The effect produced by a single oral administration of SJW on rat pain threshold was investigated in the rat paw pressure test. Administration of drugs by oral gavage can be a stressful procedure for the animal and currently it is not possible to critically evaluate the extent to which some of the drug effects observed might be due to stress-induced analgesia associated with handling and injection of rats. An orally administered vehicle group (CMC) was, therefore, included in the study. A dried extract of SJW (30 mg kg -1 p.o.) showed antinociceptive activity against a mechanical stimulus in naïve rats in comparison with CMC-treated animals, used as control group (Figure 1b). A single p.o. administration of CMC did not alter the pain threshold in comparison with the pre-treatment values (Figure 1b).

A dried extract of SJW induced a dose-dependent (1-100 mg kg -1 p.o.) mechanical antinociception also in the uninjured non-hyperalgesic leg of CCI rats. The dose of 1 mg kg -1 p.o. was devoid of any effect (F(1,14)2.714 p=0.121). At 10 mg kg -1 p.o. SJW significantly increased the rat pain threshold.

The antinociceptive effect peaked at 30 mg kg -1 p.o. and then it diminished (Figure 1a).

SJW was also able to increase the pain threshold in rats treated for 21 days with a daily injection of saline solution, animals that represented the control group of the oxaliplatin-treated rats (Figure 1d). (Figure 2a) as well as in the saline-treated group (Figure 2c): the antinociception appeared 30 min after a single acute SJW p.o. administration, peaked at 90 min and persisted up to 120 min.

Analgesic and antihyperalgesic activity of SJW components.

The chloroform fraction (CHL), representing a hyperforin rich fraction, induced antinociception when administered in a concentration corresponding to the content of hyperforin present in a 30 mg kg -1 preparation of SJW dried extract. The CHL-induced increase of the pain threshold was observed in the uninjured leg of rat with CCI (Figure 3a) as well as in naïve rats (88.8±4.7). The CHL antinociceptive dose was also endowed with antihyperalgesic activity. CHL significantly reversed the hyperalgesia in CCI and OXA rat models. The effect produced by CHL administration in CCI rats, taken as example, was reported in Figure 3a. Conversely, the methanol fraction (MET), a hypericin/flavonoid rich fraction, selectively induced antihyperalgesia without changing the baseline mechanical threshold in control rats (lack of antinociceptive properties). MET, administered in a concentration corresponding to the content of hypericin present in a 30 mg kg -1 preparation of SJW dried extract, selectively reversed the mechanical hyperalgesia in the hind paw affected by CCI (Figure 3b) as well as in the OXA model (Figure 3c) by increasing the pain threshold up to values comparable to the control ones. Time-course experiments showed that CHL and MET were endowed with the same pharmacological profile of the SJW dried extract (Figure 3a,b,c). Purified hyperforin, when administered alone, produced an antinociceptive/antihyperalgesic effect of intensity comparable to CHL fraction (Figure 4a). Similarly, purified hypericin reversed the mechanical hyperalgesia up to a value comparable to that produced by MET fraction (Figure 4b). The purified flavonoids amentoflavone (AME) and hyperoside (HypS) neither showed antinociceptive/antihyperalgesic properties (Figure 4c) nor increased hypericin-induced antihyperalgesia (Figure 4d). Quercetin (QUER) produced a significant antihyperalgesic activity even if of lower intensity than hypericin (Figure 4c), but it was unable to potentate hypericin effect

Mechanism of antinociceptive/antihyperalgesic action of SJW dried extract and components.

SJW dried extract antinociception (30 mg kg -1 p.o.) was prevented by the opioid antagonist naloxone (1 mg kg -1 i.p.) whereas the antihyperalgesic activity was only slightly reduced (Figure 5a). The increase of pain threshold as well as the prevention of the hyperalgesia produced by CHL was completely antagonized by pre-treatment with the opioid antagonist naloxone (Figure 5b).

Conversely, the antihyperalgesia induced by MET was naloxone insensitive (Figure 5c). The administration of the PKC activator PMA (15 pmol per mouse i.c.v.) significantly reduced the SJW dried extract (30 mg kg -1 p.o.) antihyperalgesic activity whereas the antinociception was only slightly reduced (Figure 6a). PMA selectively prevented the antihyperalgesic effect induced by MET (Figure 6b), but was devoid of any effect against the CHL-induced increase of pain threshold (Figure 6c). Similar results were obtained by administering purified hyperforin and hypericin (data not shown). The opioid antagonist naloxone as well as the PKC activator PMA, at the concentrations used, were devoid of any antinociceptive/hypernociceptive effect (71.7±4.7; 72.0±1.7, respectively). The dose of naloxone of 1 mg kg -1 has been demonstrated to prevent morphine analgesia without modifying the increase of the pain threshold induced by modification of other neurotransmission systems (i.e. GABAergic, muscarinic, serotoninergic, etc.). Higher naloxone doses were not employed since lacking of selectivity toward opioid analgesia.

Rats underwent CCI operation showed increased levels of PKCγ and PKCε (Figure 6d) isoforms in the PAG area. This effect was detected in the total protein preparation (TOT), in the membrane Immunoblots were re-probed for a protein considered to be not regulated as β-actin and no significant density difference was revealed for this protein among samples.

Cross-reactivity of the primary antibodies used was excluded.

Effects of SJW dried extract and fractions on rat locomotor behaviour.

The SJW dried extract, CHL and MET fractions were tested, at the highest effective doses, in order to assess their effect on rat locomotor behaviour. Rats pretreated with the above-mentioned extracts at the dose of 30 mg kg -1 p.o. were evaluated for motor coordination by use of the rota-rod test. The number of falls from the rotating rod, evaluated before and 60, 90 and 120 min after treatments, showed the lack of any impairment in the rat motor coordination by SJW dried extract and fractions administration in comparison with the control group (CMC) (Figure 7a). Each group progressively reduced its number of falls because mice learned how to balance on the rotating rod.

Amitriptyline, used as antinociceptive reference drug, at the dose of 10 mg kg -1 did not impair the motor coordination of rats. Conversely, at a 3 times higher dose (30 mg kg -1 ), it produced an increase in the number of falls from the rotating rod indicating the presence of motor incoordination (Figure 7a). SJW, at a dose of 1000 mg kg -1 was still devoid of any side effect (Figure 7a).

Similarly, purified hypericin, hyperforin, quercetin, hyperoside and amentoflavone, administered at a concentration corresponding to the content present in 30 mg kg -1 p.o. preparation of SJW dried extract, were devoid of any effect on the locomotor behaviour (Figure 7b).

A c c e p t e d

M a n u s c r i p t

Discussion

We report here the first description of the capability of a SJW dried extract to reduce hyperalgesia in rat models of neuropathic pain after a single oral administration.

Painful neuropathy is a condition that may develop when nerve fibres are damaged or dysfunctional. Since it can arise from a wide variety of injuries, two different models of neuropathic pain were used. The chronic constriction injury of the sciatic nerve was used as a model of neuropathy being induced by nerve damage whereas the repeated administration of oxaliplatin represented a chemotherapy-induced neuropathic pain model. A single oral administration of SJW reversed mechanical hyperalgesia in both models of neuropathic pain producing a prolonged increase of the pain threshold that continued up to 120 min. The efficacy of SJW in the management of neuropathic pain, regardless of the kind of injury responsible for the painful condition, was suggested. A clinical study conducted in patients suffering from painful polyneuropathy reported a trend of lower pain scores when treated with SJW than did the patients who were treated with a placebo. A statistically significant relief of pain was not obtained [START_REF] Sindrup | St. John's wort has no effect on pain polyneuropathy[END_REF], and this discrepancy might be explained by considering that the SJW-induced antinociception has a bell-shaped trend. The authors looked for an increase of the pain threshold at a concentration (2.7 mg/die total hypericins) similar to that which was employed in trials with depressed patients. This concentration was about 30 times higher than the analgesic concentration (0.096 mg total hypericins) and, as demonstrated by dose-response experiments, at this level the antinociceptive efficacy of SJW is strongly reduced.

At doses able to reverse hyperalgesia, SJW induced mechanical antinociception in untreated nonpainful rats (control groups) as well as in the controlateral uninjured hind limbs of CCI rats. These reports confirm and support the hypothesis for the role that SJW has in the modulation of pain perception. This hypothesis has been suggested in a recent paper that indicates the antinociceptive properties of SJW in the mouse formalin test [START_REF] Uchida | Antinociceptive effects of St. John's Wort, Harpagophytum Procumbens extract and grape seed proantocyanidins extract in mice[END_REF].

The main components of the SJW dried extract are phloroglucinols, naphtodiantrones and flavonoids. The effects produced by these constituents were investigated in order to discover the A c c e p t e d M a n u s c r i p t 18 components responsible for the antinociceptive activity and to better elucidate the SJW mechanism of analges ic action. We obtained a chloroform fraction (CHL) containing phloroglucinols, mainly represented by hyperforin, and a methanol fraction (MET) that was a hypericin and flavonoid rich fraction. CHL fraction reversed the mechanical hyperalgesia in all neuropathic pain models and increased the pain threshold in the control non-painful animals. The intensity of the CHL fraction activity was lower than that of the SJW dried extract, but a similar time-course profile was observed. The MET fraction was as effective as the CHL in relieving neuropathic pain. This indicates that the SJW main components present in both fractions are responsible for the antihyperalgesic properties of the plant. It is interesting to note that MET, conversely to CHL, was devoid of any analgesic activity in the non-painful rats. These results might suggest a selective role of hypericin/flavonoid in the relief of neuropathic pain, rather than being effective as a direct result of their antinociceptive properties.

The SJW antinociception underlies the activation of the opioid system as this analgesia is prevented by the opioid antagonist naloxone. Binding of [ 3 H]naloxone to the µ-and k-opioid receptor was inhibited in the presence of SJW extract [START_REF] Simmen | Hypericum perforatum inhibits the binding of mu-and kappa-opioid receptor expressed with the Semliki Forest virus system[END_REF], further supporting a SJW opioid mechanism. Phloroglucinols might be responsible for this opioid effect since hyperforin inhibited binding to the opioid receptors with a higher affinity than hypericin and flavonoids [START_REF] Simmen | Hypericum perforatum inhibits the binding of mu-and kappa-opioid receptor expressed with the Semliki Forest virus system[END_REF][START_REF] Simmen | Neurochemical studies with St. John's Wort in vitro[END_REF]. The prevention by naloxone of CHL-induced antinociception further supports this hypothesis. CHL fraction contains phloroglucinols together with a small amount of the flavonoid biapigenin that binds to the opioid receptor with a much lower affinity than hyperforin [START_REF] Simmen | Neurochemical studies with St. John's Wort in vitro[END_REF]. The administration of purified hyperforin produced an antinociceptive effect comparable to that induced by the CHL fraction, which further suggests the prominent role of hyperforin in the CHL activity.

The MET fraction contains hypericins and flavonoids, both of which might be potentially involved in the SJW mechanism of antinociceptive action. The total amount of hypericins add up to about 0.3% of the SJW dried extract and have long been known to be related to many pharmacological properties of SJW. Recent studies report antidepressive, antineoplastic, antitumor and antiviral activities of hypericin [START_REF] Kubin | Hypericin -The facts about a controversial agent[END_REF]. Flavonoids contribute to the antinflammatory effects of the A c c e p t e d M a n u s c r i p t

19 plant [START_REF] Tedeschi | Antiinflammatory actions of St. John's wort: inhibition of human inducible nitric-oxide synthase expression by down-regulating signal transducer and activator of transcription-1alpha (STAT-1 alpha) activation[END_REF] and antinociceptive properties have been reported for the flavonoid myricitrin [START_REF] Meotti | Analysis of the antinociceptive effect of the flavonoid myricitrin: evidence for a role of the L-arginine nitricoxide and protein kinase C pathways[END_REF].

Enzyme assays performed on rat brains demonstrated that hypericin and pseudohypericin are potent and selective inhibitors of the protein kinase C (PKC) [START_REF] Takahashi | Hypericin and pseudohypericin specifically inhibit protein kinase C: possible relation to their antiretroviral activity[END_REF][START_REF] Kubin | Hypericin -The facts about a controversial agent[END_REF]. In vitro studies reported that some flavonoids inhibit rat brain PKC activity. Among the flavonoids, quercetin is one of the most significant [START_REF] Ferriola | Protein kinase C inhibition by plant flavonoids[END_REF]. PKC is a family of enzymes involved in numerous important cellular events, including pain modulation. PKC integrates numerous receptor pathways into final effectors that increase excitatory signalling and decrease inhibitory signalling, thus inducing pain. The activation of PKC has been related to the induction of a painful condition whereas PKC blockers decreased nociception [START_REF] Velàzquez | Protein kinase C in pain: involvement of multiple isoforms[END_REF]. Pretreatment with the PKC activator PMA antagonized the MET-induced antinociception, whereas with the CHL-induced, the increase of the pain threshold remained unmodified. Conversely to the CHL fraction, the MET fractions mechanical antihyperalgesia remains unmodified by the naloxone pre-treatment. These results suggest that a selective blockade of the PKC-mediated intracellular pathway underlies the MET activity.

PKC is a family of serine/threonine kinases that are divided into three groups based on calcium and diacylglicerol dependence: conventional (α, ßI, ßII, γ), novel (δ,ε,η,θ), atypical (ζ, λ/ι) [START_REF] Way | Identification of PKC-isoforms-specific biological actions using pharmacological approaches[END_REF]. Among them, PKCε and PKCγ isoforms appear to play a prominent role in the modulation of pain perception [START_REF] Velàzquez | Protein kinase C in pain: involvement of multiple isoforms[END_REF][START_REF] Malmberg | Preserved acute pain and reduced neuropathic pain in mice lacking PKCγ[END_REF][START_REF] Dina | Role of protein kinase Cε and protein kinase A in a model of paclitaxel-induced painful peripheral neuropathy in the rat[END_REF]. In particular, the translocation of these enzymes from the cytosol to the synaptic membrane is thought to be necessary for their activation [START_REF] Nishizuka | Intracellular signalling by hydrolysis of phospholipids and activation of protein kinase C[END_REF]. Increased translocation and activation of PKC in dorsal horn neurons has been shown in a number of pain models. This suggests the importance in the modulation of pain perception of spinally located PKC [START_REF] Velàzquez | Protein kinase C in pain: involvement of multiple isoforms[END_REF]. We have detected a specific upregulation of PKCγ and PKCε isoforms in the PAG area of neuropathic rats along with a robust increase of the PKCγ and PKCε phosphorilation. Furthermore, we discovered a lack of increase in any of the phosphorylation of PKCε and PKCγ in the cytosol in comparison with the high phosphorilated form levels observed in the membrane fraction of neuropathic rats. These results have highlighted the importance of supraspinally located PKC in the modulation of the sensation of pain. This suggests that in the presence of neuropathies, the activation of the PKC-mediated pathway is not only confined to the spinal cord. The SJW oral To identify the MET component responsible for the increase of pain threshold, the effect produced by administration of purified MET fraction components was investigated. Hypericin showed antinociceptive properties of intensity comparable to that produced by the MET fraction.

Conversely, hyperoside, which is the most abundant flavonoid present in the SJW dried extract, neither increased the pain threshold nor potentated the hypericin-induced antinociception. Antiinflammatory properties were reported for the flavonoid amentoflavone [START_REF] Kim | Biochemical pharmacology of biflavonoids: implications for anti-inflammatory action[END_REF], but, when administered alone in neuropathic rats, it was devoid of any antinociceptive effect. Quercetin was unable to potentate hypericin antinociception when co-administered, even if it resulted in being slightly effective in increasing the pain threshold. This is not surprising since quercetin, which is similar to hypericin, has PKC-blocking properties. A quercetin potency about 30 times lower [START_REF] Ferriola | Protein kinase C inhibition by plant flavonoids[END_REF] than hypericin [START_REF] Takahashi | Hypericin and pseudohypericin specifically inhibit protein kinase C: possible relation to their antiretroviral activity[END_REF] might explain the lower efficacy observed in behavioural tests. Hypericin appears, therefore, to be the most effective component of the MET fraction.

An important drawback of the current analgesic therapy of neuropathic pain is the high occurrence of side effects [START_REF] Dworkin | Pharmacological management of neuropathic pain: evidence-based recommendations[END_REF]. The potential induction of side effects by SJW and components at the doses able to increase the pain threshold was, therefore, investigated. In accordance with the literature data that indicate SJW as a medicinal plant with a favourable tolerability and safety profile [START_REF] Linde | St John's wort for depression[END_REF], the SJW dried extract, CHL and MET fractions, at the highest active doses employed in the present study, caused neither detectable modification in the rats gross behaviour, nor did they alter their locomotive activity. Similar profile was also demonstrated by purified SJW main components.

In conclusion, our findings showed that a single oral administration of SJW, CHL and MET fractions, as well as purified hyperforin and hypericin, relieve neuropathic pain in rats with a very A c c e p t e d M a n u s c r i p t 21 positive side effects profile. This increase of the pain threshold is related to the modulation of two different intracellular pathways: the activation of the opioid system, underling the presence of hyperforin, which is responsible for the antinociceptive properties, and a PKC-mediated mechanism through the PKC-blocking properties of hypericin, mainly related to the antihyperalgesic activity.

Finally, these results further support the hypothesis that the inhibition of PKC specific isoforms might be a promising strategy for effective treatment of neuropathic pain.

ACKNOWLEDGEMENTS:

This work was supported by grants from MIUR.

  glucose solution. The experiments were carried out on day 21.

A c c e p t e d M a n u s c r i p t 10 perforatum

 10 dried extract -Lotto 28662/M1 -B.A. 84204 and kindly provided the reference rutin trihydrate (batch no. K12408717, standard purity 96.25% considering the content of residual solvents, moisture and amount of impurities).

A c c e p t e d M a n u s c r i p t 11 Naloxone ( 1

 111 mg kg -1 i.p.) was injected 70 min after SJW, CHL or MET administration. PMA (15 pmol per mouse i.c.v.) was injected 30 min after SJW, CHL or MET administration.

A c c e p t e d M a n u s c r i p t 13 1d

 13

A c c e p t e d M a n u s c r i p t 14 Time

 14 course experiments confirmed the prolonged effect of SJW in the uninjured leg of CCI rats

A c c e p t e d M a n u s c r i p t 15 (

 15 Figure4d).

(

  MEM) and cytosol (CYT) fractions. A robust increase of the phosphorylation of PKCγ and PKCε was also observed in the total proteins (TOT) (PKCγ: F(1,10)15.536 p<0.001; PKCε: F(1,10)18.196 p<0.001) and in the membrane fraction (MEM) whereas in the cytosol fraction (CYT) the levels of p-PKCγ and p-PKCε were similar to the control group (Figure 6d). SJW treatment was able to A c c e p t e d M a n u s c r i p t 16 reduce the phosphorilation of both PKC isoforms as demonstrated by the decrease of p-PKCγ and p-PKCε levels up to values corresponding to the control (Figure 6d). Similar results were obtained in the PAG of OXA rats (data not shown).

A c c e p t e d M a n u s c r i p t 20 administration

 20 produced a complete reversal of the phosphorylation of both PKCε and PKCγ in the total protein preparation as well as in the membrane fraction. These data suggest the hypothesis of a PKC-inhibiting mechanism in SJW for antinociception and indicate the isoforms ε and γ of PKC as potential targets for this antinociceptive effect. Furthermore, the importance of the inhibition of the PKC activity in order to treat neuropathic pain is further confirmed.

Figure 1 .

 1 Figure 1. Dose-response curves for the antinociceptive and antihyperalgesic effect of SJW dried extract in the chronic constriction injury of the sciatic nerve (CCI) and repeated oxaliplatin treatment (OXA) rat model of neuropathic pain. (a) Dose-response curve of SJW dried extract (1-100 mg kg -1 p.o.) for the antinociceptive (uninjured leg; SJW 10 mg kg -1 p.o. (F(1,22)6.730 p<0.05; SJW 30 mg kg -1 p.o. (F(1,17)35.317 p<0.001) and antihyperalgesic (injured leg; SJW 30 mg kg -1 p.o. F(4,44)10.690 p<0.001) effect in the CCI rats. Data were recorded 90 min after SJW administration. Experiments were performed 14 days after surgery when a significant mechanical hyperalgesia was obtained (F(1,19)44.631 p<0.001) *P<0.001 in comparison with values recorded before treatment in the corresponding leg; ^P<0.001 in comparison with the uninjured leg. (b) Time-course curve of SJW dried extract (30 mg kg -1 p.o.) for the antinociceptive activity in naïve rats. *P<0.01 in comparison with CMC-treated group. (c) Time-course curve of the mechanical hyperalgesia induced by repeated administration of oxaliplatin in comparison with saline-treated rats. A statistically significant hyperalgesia appeared on day 21 (F(1,14)22.345 p<0.001); *P<0.001 compared to saline control group. (d) Dose-response curve of SJW dried extract (10-100 mg kg -1 p.o.) for the antinociceptive (saline-treated group; SAL) and antihyperalgesic (OXA; F(4,42)12.718 p<0.001) effect in the rat OXA model of neuropathic pain. Data were recorded 90 min after SJW administration. *P<0.001 in comparison with corresponding values recorded before treatment, ^P<0.001 compared to saline control group. The experiments were carried out on day 21.

A c c e p t e d M a n u s c r i p t

Figure 2 .

 2 Figure 2. Time-course curves for the antinociceptive and antihyperalgesic effect of SJW dried extract in rat models of neuropathic pain induced by chronic constriction injury of the sciatic nerve (CCI) and repeated administration of oxaliplatin (OXA). Time-course curves of SJW dried extract (30 mg kg -1 p.o.) in the CCI model for the antinociceptive (uninjured leg) (a) and antihyperalgesic (injured leg) (b) effect. *P<0.05 in comparison with CMC-treated group. All data are presented as mean ± s.e.m. (c) Time-course curves of SJW dried extract (30 mg kg -1 p.o.) in the OXA model for the antinociceptive (SAL) and antihyperalgesic (OXA) effect. *P<0.05 in comparison with SAL before treatment; ^P<0.05 in comparison with OXA before treatment. All data are presented as mean ± s.e.m. The experiments were carried out on day 21.

A c c e p t e d M a n u s c r i p tFigure 3 .

 3 Figure 3. Antinociceptive profile of SJW fractions containing the SJW major components in rat models of neuropathic pain. (a) Antinociceptive (uninjured leg; F(3,35)5.422 p<0.001) and antihyperalgesic (injured leg) effect of the chloroform (CHL) fraction, administered in an amount containing 0.978 mg kg -1 hyperforin, in the CCI model. Data are presented as mean ± s.e.m.; *P<0.001 in comparison with the uninjured leg before treatment; ^P<0.01 in comparison with the injured leg before treatment. (b) Antihyperalgesic (injured leg; F(3,32) 6.002 p<0.001) effect of the methanol (MET) fraction (30 mg kg -1 ), administered in an amount containing 0.06 mg kg -1 hypericin, in the CCI model. Data are presented as mean ± s.e.m.; *P<0.001 in comparison with uninjured leg before treatment; ^P<0.05 in comparison with injured leg before treatment. (c) MET prevented the mechanical hyperalgesia induced by oxaliplatin (OXA; F(3,33) 7.244 p<0.001) administration without inducing antinociception in the saline (SAL) group. Data are presented as mean ± s.e.m.; *P<0.001 in comparison with SAL; ^P<0.001 in comparison with OXA. The experiments were carried out on day 21.

A c c e p t e d M a n u s c r i p t 29 Figure 4 .

 294 Figure 4. Antinociceptive profile of purified components of CHL and MET fractions in the CCI model of neuropathic pain. (a) Purified hyperforin (0.978 mg kg -1 ) showed antinociceptive (uninjured leg) and antihyperalgesic (injured leg) properties with a similar profile to CHL (F(3,32)4.871 p<0.001). Data are presented as mean ± s.e.m.; *P<0.001 in comparison with uninjured leg before treatment; ^P<0.05 in comparison with injured leg before treatment. (b) Hypericin (0.06 mg kg -1 ), similarly to MET, reversed the mechanical hyperalgesia (injured leg; F(3,35)3.992 p<0.001) without increasing the pain threshold in the non-hyperalgesic (uninjured) leg. Data are presented as mean ± s.e.m.; *P<0.001 in comparison with uninjured leg before treatment; ^P<0.001 in comparison with injured leg before treatment. (c) Lack of effect on pain threshold of the purified flavonoids amentoflavone (AME, 0.016 mg kg -1 ) and hyperoside (HypS, 1.905 mg kg -1 ). Quercetin (QUER, 0.249 mg kg -1 ) showed a modest antihyperalgesic effect (F(1,14)3.071 p<0.05). Data are presented as mean ± s.e.m.; Data were recorded 90 min after administration of SJW constituents. *P<0.05 in comparison with uninjured leg before treatment; ^P<0.01 in comparison with injured leg before treatment. (d) Amentoflavone (AME), hyperoside (HypS) and quercetin (QUER) were unable to potentate the hypericin-induced antihyperalgesic effect. Data were recorded 90 min after administration of SJW constituents. Data are presented as mean ± s.e.m. *P<0.01 in comparison with uninjured leg before treatment; ^P<0.05 in comparison with injured leg before treatment. Each component was administered at a dose corresponding to the amount of each component present in a 30 mg kg -1 preparation of SJW dried extract.

A c c e p t e d M a n u s c r i p t 30 Figure 5 .

 305 Figure 5. Involvement of the opioid system in the antinociceptive activity of SJW and CHL fraction in the CCI model of neuropathic pain. (a) Prevention by naloxone (1 mg kg -1 i.p.) of SJW

A c c e p t e d M a n u s c r i p tFigure 6 .

 6 Figure 6. Blockade of the PKC activity as mechanism of the antihyperalgesic effect of SJW and MET fraction in the CCI model of neuropathic pain. (a) The administration of the PKC activator PMA (15 pmol per mouse i.c.v.) significantly reduced the SJW dried extract (30 mg kg -1 p.o.) antihyperalgesic activity (F(1,14)5.729 p<0.001) whereas the antinociception was only slightly reduced (F(1,14)2.714 p=0.0487). Data were recorded 90 min after administration of SJW. (b) Prevention by the PKC activator PMA of MET antihyperalgesic activity (injured leg; F(1,15)6.231 p<0.001). Data were recorded 90 min after administration of MET. (c) Lack of effect of PMA on the CHL-induced antinociception (uninjured leg) and antihyperalgesia (injured leg). Data were recorded 90 min after administration of CHL. Data are presented as mean ± s.e.m.; *P<0.001 in comparison with uninjured leg; ^P<0.01 in comparison with injured leg. (d) The CCI induced an increase in the PKCγ and PKCε isoforms levels observed in the total protein preparation (TOT; PKCγ: F(1,10)3.991 p<0.01; PKCε: F(1,10)4.961 p<0.01) and in the membrane (MEM) and cytosol (CYT) fractions from the periaqueductal grey (PAG) area of neuropathic rats. A dramatic increase of the phosphorylation of PKCγ and PKCε was observed in the total protein preparation (TOT) and membrane (MEM) fraction whereas in the cytosol (CYT) fraction no variation was detected. SJW decreased the phosphorylation of PKCγ and PKCε isoforms up to control values. Densitometric values are reported as percentage of control. *P<0.01 in comparison with control group; §P<0.05 in comparison with CCI.

A c c e p t e d M a n u s c r i p t

Figure 7 .

 7 Figure 7. Lack of induction of locomotor side effects by SJW dried extract, fractions and purified components. (a) Lack of effect of SJW dried extract, CHL and MET fractions on rat motor coordination evaluated in the rat rota rod test. Effects produced by amitriptyline (AMI) at the doses of 10 and 30 mg kg -1 , used as reference antinociceptive drug. *P< 0.05 in comparison with CMCtreated rats. (b) Purified SJW main components showed the lack of any influence on locomotor behaviour. A total of 4-5 rats per group were tested.

  pain in different rat models through a decrease of PKC  and  isoform expression and phosphorylation.

  Figure

  Figure

  -1 i.p.) of SJW

	dried extract (30 mg kg -1 p.o.) antinociception (naïve, sham-operated, uninjured leg; F(1,18)9.487
	p<0.001). Lack of effect on the antihyperalgesic activity (injured leg; F(1,14)2.714 p=0.121). Data
	were recorded 90 min after administration of SJW. Data are presented as mean ± s.e.m.; *P<0.001
	in comparison with uninjured leg before treatment; ^P<0.01 in comparison with injured leg before
	treatment. (b) Prevention by naloxone of CHL (administered in a dose containing 0.978 mg kg -
	1 hyperforin) antinociception (naïve, sham-operated, uninjured leg; F(1,16)8.422 p<0.001) and
	antihyperalgesia (injured leg; F(1,16)5.612 p<0.01). Data were recorded 90 min after administration
	of CHL. Data are presented as mean ± s.e.m.; *P<0.05 in comparison with uninjured leg before

treatment; ^P<0.05 in comparison with injured leg before treatment. (c) Lack of effect of naloxone on MET (administered in a dose containing 0.06 mg kg -1 hypericin) antihyperalgesia (injured leg). Data were recorded 90 min after administration of MET. Data are presented as mean ± s.e.m.; *P<0.05 in comparison with uninjured leg before treatment; ^P<0.05 in comparison with injured leg before treatment.

Table 1 . Composition of the SJW dried extract, CHL and MET fractions Content % (mg/100 mg)

 1

	Constituent

CHL: chloroform fraction; MET: methanol fraction. The content of each constituent is expressed as percentage (mg/100 mg) Table A c c e p t e d


Alberto Zafra-Gómez

Juan Carlos Morales

Oscar Antonio Ballesteros

Alberto Navalón

A Sensitive Gas Chromatographic-Mass Spectrometric (C-MS) Method for the Determination of Bisphenol A in Rice-Prepared Dishes

Keywords: Bisphenol A, Migration, Gas chromatography-mass spectrometry (GC-MS)

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

BPA is a compound with high reactivity and is the raw material for a large amount of 25 manufactured products, such as epoxy resin, or polysulfones. It is also used as an 26 antioxidant or stabilizer. However, one of the most important applications of the compound 27 is the production of polycarbonate plastics, which are mainly a condensed polymer of BPA 28 and carbonyl chloride or diphenyl carbonate. Since it is transparent, has excellent heat 29 resistance and impact resistance, and can be used for high-temperature uses and in 30 microwave ovens, it is used in items such as children's tableware, coffee makers and food 3 Some studies have reported that BPA has an estrogen-like action of toxicity; 1 moreover, it was discovered that the compound was released from a flask made of 2 polycarbonate and showed binding to the estrogenic receptor. In fact, BPA induced 3 progesterone receptors in cultured breast cancer cells and its activity was about 1/5,000 4 that of estradiol (Krishnan et al., 1993). Different studies show statistically significant Prior to extraction, sample was carefully homogenized with a mixer and sample (20 g) 14 were placed in 50 mL falcon tube. After that, te sample was spiked with bisphenol F as a 15 surrogate (100 ng•g -1 ). Extraction was carried by adding 5 mL of acetonitrile and shaking 16 vigorously in a vortex-mixer for 1 min. The mixture was centrifuged 2 min at 6000 r.p.m. 17 at room temperature. Supernatant was transferred into a 25 mL separation funnel, and the 18 extraction was repeated twice. NaCl (5 g) were added to the combined organic phase in the 19 separation funnel and three phases, entailing a solid sodium chloride phase, saturated NaCl 20 aqueous solution phase and an organic phase, could be observed. After removing the solid 21 and the aqueous layer, the acetonitrile phase was washed with n-hexane (2 mL) to remove 22 fat traces and decanted into a 10 mL assay tube in order to evaporate to dryness in a speed-23 vac system. The extract was re-dissolved using 1.0 mL of ethyl acetate and transferred into 24 a chromatographic vial.

25

After evaporation to dryness under nitrogen, 50 µL of a mixture of ethyl 26 acetate/BSTFA/Pyridine (2:1:1; v/v/v) were added into the tube to resuspend the residue 27 and to carry out the derivatization. BPA and BPF are suitable for derivatization due to their 28 chemical structure. Once the derivatization process was completed (1 minute), 1 µL of the 29 reaction mixture was injected into the GC-MS system. The GC oven temperature program applied was as follows: the initial oven temperature 11 was set at 150 ºC, held for 0.5 min, then the temperature was increased to 280 ºC via ramp 12 of 20 ºC min -1 and held for 5.0 min, finally the temperature was raised to 300ºC at 40 ºC 13 min -1 , and maintained for 2.5 min. The total run time was 15.0 min. The carrier gas used 14 was helium (purity 99.999%) at a flow rate of 1.0 mL min -1 . Delay time was 3 min in order 15 protect the ion multiplier of the MS instrument from saturation and the sample volume in 16 the direct injection mode was 1 µL. The BSTFA/Pyridine concentration was studied from 0 to 100%, temperature from 25 2 (room temperature) to 95 ºC and the reaction time was varied between 0 and 60 min. A 3 50% of silylation agent in ethyl acetate (v:v), 1 min and room temperature were the 4 procedural conditions selected. Calibration graphs for samples treated according to the analytical procedure described 5 above were made using SIM mode. BPF was used as surrogate.

6

The standard addition calibration was carried out by fortification of homogenized rice 7 samples aliquots. 20 g of raw samples free of BPA were placed in a 50 mL falcon tubes 8 collection. Then, samples were spiked with BPF as a surrogate (100 ng•g -1 ) and with BPA 9 as analyte at growing concentrations. The standard samples were strongly shaken in order 10 to mix surrogate, analyte and sample and treated as described in the "Extraction and 11 derivatization" section. Linearity of the calibration using standard addition graphs was 12 tested according to the Analytical Methods Committee (Analytical Methods Committee, 13 1994).The lack-of-fit test was applied to the residuals of a calibration curve. Two Linearity. A concentration range from the minimal amount detectable by this 25 methodology up to two orders of magnitude higher (0.X to 100 ng g -1 ) was selected for 26 method application.

27

The response of compound was checked in the range of application of the analytical 28 method by linear regression analysis by the least-squares method of peak area ratio of 29 analyte/surrogate against different analyte concentrations. The response was linear in the 30 range of concentrations evaluated.

31

In the specific case of higher BPA concentration than the selected range, we should 32 test the linearity of method beyond 100 ng g -1 or simply dilute the corresponding samples 33 if necessary. In our application, no levels higher than 100 ng g -1 were found. The recoveries for BPA were between 90% and 105 % in all cases as is shown in Table 3. A fundamental aspect which needs to be examined in the validation of any 22 analytical method is its limit of detection in order to determine if an analyte is present in 23 the sample. In this work, a criterion for method performance has been used that includes 24 the decision limit, CC α , and the detection capability, CC β (COMMISSION DECISION, 25 2002). Decision limit (CCα) is defined as the limit at and above which it can be concluded 26 with an error probability of α that a sample is non-compliant. Detection capability (CC β ) is 27 defined as the smallest content of the substance that may be detected, identified and/or 28 quantified in a sample with an error probability of β. They were calculated according to the better adjusted to a statistical evaluation are implemented. Thus, CC α (α = 5%) and CC β (β 1 = 5%) are also summarized in Table 2. 2 Selectivity. Compound was quantified using selected ion recording mode (SIM).

3 Analyte appear to be well resolved and free from interference peaks (Figure 1). The 4 identity of the chromatographic peak was confirmed not only by its retention time but also 5 by its mass spectrum (3 fragments were used). The method has been used routinely for monitoring the presence of BPA in the production 27 of rice-prepared dishes and no contamination has been found.

28

Our results indicate that the levels of BPA identified in rice-prepared dishes are 29 unlikely to be of concern to adult health, and there is no reason for consumers to change 30 their consumption patterns as a result of these findings.

  laboratory studies describing, in some cases, severe 9 disturbances in the endocrine system of humans and wildlife have been published in the 10 past few years (Rivas et al., 1997; Paris, et al., 2002). Today, it is well known that around 11 100 industrial chemicals show estrogenic activity in addition to their desired chemical 12 properties. Most of these endocrine disrupter chemicals (EDC's) are widely used organic 13 compounds, which are ubiquitous in the environment and in biological samples (Fromme 14 et al., 2002; Harris et al., 1997). At the same time, ECD's accumulate in certain tissues in 15 humans and their effects could be passed to future generations via placenta and/or milk 16 (Maffini et al., 2006; Hood, 2005; Lopez-Espinosa et al. 2008) 17 Most of these compounds, commonly called endocrine disrupter chemicals (EDCs), 18 are synthetic organic chemicals introduced into the environment by way of anthropogenic 19 inputs. Aware of the problem, both the European Union and the United States (EPA) have 20 authored a "priority" list of substances for further evaluation of their role in endocrine 21 disruption (Groshart and Okkerman, 2000; Multi-Year Plan [FY2000-2012] for Endocrine 22 Disruptor) and indicate the need to assess the levels and effects of EDCs. 23 One of the representative compounds of the group of EDC's is bisphenol A (BPA).

  24

5

  reproductive and developmental toxicity in rats and mice at high doses of BPA (Morrissey 6 et al., 1987). Nevertheless, there still controversy over whether low doses of BPA may 7 cause reproductive and developmental effects in humans (Goodman et al. 2006). 8 Significant published data is available on BADGE or BPA migration from can coatings 9 into foods and food simulants (Biles et al., 1997; Kawamura et al., 1999; Yoshida et al., 10 2001; Goodson et al., 2002; Kang and Kondo, 2003; Cabado et al., 2008) but little is 11 known about how the migration level would be influenced by damage to the can or storage 12 conditions. However, some workers have investigated the effects of the food processing 13 conditions on the migration of BPA from food or from simulating liquids (Munguia-Lopez 14 and Soto-Valdez, 2001, Sajiki et al., 2005; Le et al, 2008; Poças and Hogg, 2007; Mungia-15 Lopez et al., 2002). 16 Sterilization conditions, normally applied by industry for the scenarios used in our 17 study are relatively extreme (high temperature and sterilization time). In view of the 18 potentially long shelf-life of most canned foods and the current interest surrounding the 19 exposure to BPA from food packaging, it was considered important to obtain information 20 on all of these factors. 21 The Scientific Committee for Food reviewed the Tolerable Daily Intake (TDI) for 22 BPA and the TDI reduced its value to a temporary one of 0.01mg kg -1 body weight day per 23 day. Therefore, it is of crucial importance to devise analytical methodology for detecting 24 and quantifying these compounds in food. 25 Gas chromatography-mass spectrometry has traditionally been used as the main 26 analytical methodology for analyzing BPA together with other EDCs (nonylphenol, 27 phthlates or bisphenol A diglycidyl ether) in water, milk or beverages (Mol et al., 2000; 28 Rodríguez et al., 2003; Helaleh et al., 2001; Ding and Chiang, 2003; 2000; Schoene et al., 29 1994; Nakamura et al., 2001; Fine et al., 2003; Casajuana and Lacorte, 2004;). In contrast, 30 there are very few examples where GC-MS has been applied to powdered (Kuo and Ding, 31 2004) or solid foods (Thomson and Grounds, 2005). Goodson et al., (2004) investigated 32 the potential effects, on the migration of BPA from can coatings, of cooking or heating 33 foods in the can prior to consumption using GC-MS as their analytical technique.Samples were randomly selected from different supermarkets and also from a rice-1 prepared dishes production plant situated in Seville (Spain). A total of 250 different rice-2 prepared dishes were acquired and analyzed.3As well, rice-prepared samples were collected previous to the packing procedure 4 directly in the production plant. These samples were homogenized and tested as negative 5 controls for BPA and, at the same time, were used for fortification and/or recovery studies. 6 All samples were carefully mixed and homogenized using a food mixer. The 7 homogenized samples were stored in glass bottles, previously cleaned with nitric acid (1:1; 8 v/v), in the dark and at 4 ºC until treatment. The analysis was performed with the minimum 9 possible delay and as described below in the Extraction and derivatization section in order 10 to avoid sample degradation. 11 12 Extraction and derivatization 13

  performed using an Agilent 6890 Series GS System GC fitted with a 1 splitless injector for a low background, a split/splitless deactivated glass liner (78.5 mm × 2 6.3 mm × 4.0 mm, wool packed) for capillary injection port was used. A J&W Scientific 3 Inc capillary column DB-5MS fused-silica, 30 m × 0.25 mm I.D.; 0.25 µm film thickness; 4 5% phenyl-95% dimethyl arylene siloxane was used. Detection was carried out with a 5 5973 mass-selective single quadrupole detector (Agilent technologies). The GC-MS 6 operation control and the data process were carried out by ChemStation software. 7 The injector port of the GC was set at 250 ºC. The silylated samples were 8 automatically injected using the splitless-injection mode. The transfer line of the GC to the 9 MS was set at 280 ºC, and the electron ionisation (EI) ion source of the MS set at 250 ºC.10

  17

For

  qualitative analysis, the MSD was operated in full-scan mode. The conditions for 18 electron impact ionization (EI) were: ion energy of 70 eV, multiplier 1800 and the mass 19 range scanned was 50-400 m/z. The MS was tuned everyday to m/z 69, 219 and 502 with 20 perfluorotributylamine (PFTBA) as a calibration standard. 21 For quantitative analysis, single ion monitoring (SIM) acquisition mode (dwell time 22 100 ms/ion) was used. The retention times for BPF and BPA trimethylsilyl derivatives 23 were 8.9 min and 10.7 min, respectively. The mass spectrum of silylated BPF showed a 24 base peak at 344 m/z, corresponding to the molecular ion, and the mass spectrum of 25 silylated BPA showed the molecular ion peak at 372 m/z, whereas the base peak appears at 26 357 m/z, corresponding to the loss of a methyl group 27 28 Statgraphics Centurion XV, vs 15.1.02 software package (1982-2006 Statpoint Inc) 29 was used for the statistical analysis of data.Isolation and pre-concentration procedure 1 Rice-prepared dishes contain a complex mixture of a large number of components 2 (rice, meat, peas, oil, fat, etc.). Determinations at the trace level require isolation and a pre-3 concentration step of the samples to reach nanogram levels of concentration. An extraction 4 procedure was selected as appropriate to obtain the analyte from the homogenized rice dish 5 samples. The extraction was optimized by adjusting parameters that influence in analyte 6 extraction, e.g., nature of extraction solvent, extraction time or cleaning solvent. 7 A large number of mixtures of diethyl ether, methanol and acetonitrile were tested for 8 extraction of analytes from samples. Pure solvents and different mixtures of diethyl ether, 9 methanol and acetonitrile (ratios 3:1, 1:1 and 1:3) were tested for extraction of analytes 10 from samples. Extraction yield for analyte isolation (at 100 ng g -1 spiking samples) was in 11 all cases between 60% and 90%, except for pure acetonitrile that showed the highest yield 12 (96%) 13 In addition, n-pentane, n-hexane and chlorinated solvents (dichloromethane, 14 chloroform or carbon tetrachloride) were tested to remove fat traces from acetonitrile 15 extracts in separating funnel. Although carbon tetrachloride and chloroform showed good 16 results (close to 90% of de-fating ratio, and less than 15 % of analyte losses), they were not 17 used due to their high toxicity for humans and the environment. n-Pentane showed a 18 relative defating ratio very close to n-hexane but the analyte loss was 40% for pentane and 19 only 12 % for n-hexane. Therefore, acetonitrile was selected for BPA extraction and n-20 hexane was used for extract cleaning. 21 22 Derivatization method 23 Trimethylsilyl derivatives of BPA and BPF were obtained using a BSTFA/Pyridine 24 mixture as silylation reagent. This reagent was selected because of its fast reactivity with 25 compounds containing hydroxyl groups, its high volatility resulting in no co-elution of 26 early eluting peaks, and low thermal degradation and good solubility of the derivatized 27 compounds in common organic solvents. Derivatized samples had a better separation of 28 the analyte under GC-MS analysis, because of their higher volatility and lower interaction 29 with the stationary phase. 30 The optimisation of the derivatization procedure was carried out by applying the 31 experimental design methodology. The effect of varying the percentage of silylation agent 32 (BSTFA/PYRIDINE 1:1, v/v) in ethyl acetate, temperature of process and reaction time 33 were tested on the analytical response. The three factors were simultaneously optimised by 34 of a 2 3 central composite design plus face centred (with three centred points).1

5 6Food

 5 Figure1

  14 experimental replicates and three injections of each standard were carried out. The results 15 for the intercept (a), slope (b), correlation coefficient (R 2 ) and probability level of the lack-16 of-fit test, P lof (%), are summarized in Table 2. according to the U.S. Food and Drug Administration (FDA) 21 guideline for bioanalytical assay validation (U.S. Department of Health and Human 22 Services, 2001). The analytical performance parameters assessed for the overall assay were 23 linearity, precision, accuracy, sensitivity, and selectivity.

  24

  The precision expressed as relative standard deviation (RSD), at three 1 concentration levels, was obtained from thirty replicates of spiked samples obtained from 2 10 aliquots of a homogenate of 10 different rice dishes and analyzed during the same day 3 (repeatability) and in three different days (reproducibility). A total of 30 analyses were 4 carried out in order to calculate precision. The relative standard deviation is lower than 5 10% in all cases as is shown in Table3. Data indicate that the analytical method is precise 6 (repeatable and reproducible). It is important to note that the precision varied with 7 concentration. Important factors such as sample weight, standard preparation, instrument 8 response or calibration uncertainty can limit precision. 9 Accuracy. A recovery assay was performed by comparing the analytical results for 10 extracted samples, free of BPA, spiked at three concentration levels. The concentration of 11 the compound was determined by interpolation in the standard addition calibration curve 12 within the linear dynamic range and compared with the added amount. Ten replicates, by 13 spiking 20g of rice samples with the analyte, at three concentration levels were analyzed.

  14

  quite good considering the amount of sample, and the low 19 concentration of the analyte and complexity of the sample. The values indicate that 20 compounds are quantitatively extracted. 21 Sensitivity.

Food

  29 calibration curve procedure described in commission decision of 12 August 2002 30 implementing Council Directive 96/23/EC concerning the performance of analytical 31 methods and the interpretation of results. Decision limit and detection capacity which are 32 Page 10 of 24 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk

  has been applied as a routine method for the determination of 9 BPA in prepared rice dishes packed in plastic containers from different brands 10 commercialized in Spain. These containers are manufactured using different plastic 11 materials which could include polycarbonate plastics. 250 samples of rice-prepared dishes, 12 picked up from different points were analyzed. An example of the chromatogram obtained 13 in SCAN mode and in SIM mode for a natural sample is included in Figure 1 (C and D).

  14BPA does not seem to be present above the reported lower detection limits proposed in this shows that BPA can be detected and quantified reliably in rice-19 prepared dishes combining GC-MS with a solid-liquid extraction procedure. The assay 20 involves a preconcentration and the removal of interferences step in conjunction with a 21 silyl-derivatization procedure previous to CG-MS analysis. The GC-MS analytical method 22 developed in this study has been shown to be reliable and had a low limit of detection for 23 BPA, and has been successfully applied to spiked and non-spiked rice samples. Proper 24 sample collection in conjunction with sound storage practice prior to analysis allows for 25 good recovery values in all cases as demonstrated by the validation procedure employed.26

FoodResearch

  .E., MCNEAL, T.P., BEGLEY, T.H., 1997, Determination of Bisphenol A 6 Migrating from Epoxy Can Coatings to Infant Formula Liquid Concentrates. J. Agric. 7 .G., ALDEA, S., PORRO, C., OJEA, G., LAGO, J., SOBRADO, C., 10 VIEITES, J.M., 2008, Migration of BADGE (bisphenol A diglycidyl-ether) and 11 BFDGE (bisphenol F diglycidyl-ether) in canned food. Food Chem. Toxic., 46, 1674-. AND LACORTE, S., 2004, New Methodology for the Determination of 15 Phthalate Esters, Bisphenol A, Bisphenol A Diglycidyl Ether, and Nonylphenol in 16 Commercial Whole Milk Samples. J. Agric. Food Chem., 52 (12), 3702-3707 17 18 COMMISSION DECISION of 12 August 2002 implementing Council Directive 96/23/EC 19 concerning the performance of analytical methods and the interpretation of results 20 (notified under document number C(2002) 3044) (2002/657/EC) 21 22 DING, W.H., CHIANG, C.C., 2003, Derivatization procedures for the detection of 23 estrogenic chemicals by gas chromatography/mass spectrometry. Rapid Commun. .D. BREIDENBACH, G.P. PRICE, T.L. HUTCHINS, S.R., 2003, Quantitation of 28 estrogens in ground water and swine lagoon samples using solid-phase extraction, 29 pentafluorobenzyl/trimethylsilyl derivatizations and gas chromatography-negative 30 ion chemical ionization tandem mass spectrometry. J. Chromatogr. A, 1017 (1., UCHLER, TH. K., OTTO, TH., PILZ, K., ULLER, J.M., WENZEL A., 1 2002, Occurrence of phthalates and bisphenol A and F in the environment. Water 2 . E., McCONELL, E. E., SIPES, I. G., WITORSCH, R. J., SLAYTON, T. 5 M., YU, C. J., LEWIS, A. S., RHOMBERG, L. R., 2006, An updated weight of the 6 evidence evaluation of reproductive and developmental effects of low doses of 7 bisphenol A. Crit Rev Toxicol., 36(5), 387-457. 8 9 GOODSON, A., ROBIN, H., SUMMERFIELD, W., COOPER, I., 2004, Migration of 10 bisphenol A from can coatings-effects of damage, Storage conditions and heating. 11 Food Addit. Contam., 21, 1015-1026. 12 13 GOODSON, A., SUMMERFIELD, W., COOPER, I., 2002, Survey of bisphenol A and 14 bisphenol F in canned foods. Food Addit. Contam., 19 (8), 796-802. 15 16 GROSHART, C.H., OKKERMAN P.C. (BKH, Consulting Engineers), 2000, European 17 Commission DG ENV. Inform M0355008/1786Q/10/11/00. pp. 35. 18 19 HARRIS, C.A., HENTTU, P., PARKER, M.G., SUMPTER, J.P., 1997, The estrogenic 20 activity of phthalate esters in vitro. Environ. Health Perspect., 105(8), 802-811. 21 22 HELALEH, M.I.H., TAKABAYASHI, Y., FUJII, S., KORENANGA, T., 2001, Gas 23 chromatographic-mass spectrometric method for separation and detection of 24 endocrine disruptors from environmental water samples. Anal. Chim. Acta., 428 (. Environews NIEHS News 2005. Growth Spurt for EDC Recognition. Environ. 28 Health Perspect., 113(8), A522-A524. 29 30 KANG, J., KONDO, F., 2003, Determination of bisphenol A in milk and dairy products by 31 high-performance liquid chromatography with fluorescence detection. J. Food Prot., 32 66 (8), 1439-1443. ., SASAKI, T., MATSUDA, Y., KANEKO, S., IWAMOTO, T., 1 TANAKA, M., 2001, Sensitive determination of bisphenol A and alkylphenols by 2 high performance liquid chromatography with pre-column derivatization with 2-(4-3 carboxyphenyl)-5,6-dimethylbenzimidazole. Biomed. Chromatogr., 15 (6), 403-407. 4 5 KAWAMURA, Y., SANO, H., YAMADA, T., 1999, Migration of bisphenol A from can 6 coating to drinks. J. Food Hyg. Soc. Jpn., 40, 158-165. 7 8 KELLY, C., 2000, Analysis of steroids in environmental water samples using solid-phase 9 extraction and ion-trap gas chromatography-mass spectrometry and gas 10 chromatography-tandem mass spectrometry. J. Chromatogr. A, 872 (1-2), 309-314. 11 12 KRISHNAN, A.V., STRATHIS, P., PERMUTH, S.F., TOKES, L., FELDMAN, D., 1993, 13 Bisphenol-A: an estrogenic substance is released from polycarbonate flasks during 14 autoclaving. Endocrinol. 132, 2279-2286 15 16 KUO H.W. AND DING W.H. 2004, Trace determination of bisphenol A and 17 phytoestrogens in infant formula powders by gas chromatography-mass 18 spectrometry. J. Chromatogr. A, 1027 (1-2), 67-74. 19 20 LE, H.H., CARLSON, E.M., CHUA, J.P., BELCHER, S.M., 2008, Bisphenol A is released 21 from polycarbonate drinking bottles and mimics the neurotoxic actions of estrogen in 22 developing cerebellar neurons. Toxicology Letters, 176 (2), 149-156. 23 24 LOPEZ-ESPINOSA, M.J., KIVIRANTA, H., ARAQUE, P., RUOKOJÄRVI, P., 25 MOLINA-MOLINA, J.M., FERNANDEZ, M.F., VARTIAINEN, T., OLEA, N. 26 2008. Dioxins in adipose tissue of women in Southern Spain. Chemosphere. 73(., RUBIN, B.S., SONNENSCHEIN, C., SOTO, A.M. 2006. Endocrine 30 disruptors and reproductive health: The case of bisphenol A Molecular and Cellular 31 Endocrinology, 25, 179-186. C., MAKRI, A., LAMPI, E.N., THOMAIDIS N.S. KOUPPARIS, M.A., 1 2008, Migration of bisphenol A from polycarbonate baby bottles under real use 2 conditions. Food Add. Cont. 25(3), 373-383. 3 4 MOL, H.G.J., SUNARTO, S., STEIJGER, OM., 2000, Determination of endocrine 5 disruptors in water after derivatization with N-methyl-N-(tert.-butyldimethyl 6 trifluoroacetamide) using gas chromatography with mass spectrometric detection. J. .E., GEORGE, J.D., PRICE, C.J., TYL, R.W., MARR, M.C., KIMMEL, 10 C., 1987, The developmental toxicity of bisphenol A in rats and mice. Fundam. Appl. 11 Toxicol., 8, 571-582. 12 13 MULTI-YEAR PLAN (FY2000-2012) for Endocrine Disruptor. Office of Research and 14 Development. US EPA. December 2003. http://www.epa.gov/osp/myp/edc.pdf 15 16 MUNGUIA-LOPEZ, E., SOTO-VALDEZ, H., 2001, Effect of Heat Processing and 17 Storage Time on Migration of BPA and BADGE to Aqueous Food Simulant from 18 Mexican Can Coatings. J. Agric. Food Chem. 42 (8). 1497-1501. 19 20 MUNGUÍA-LÓPEZ, E. M., GERARDO-LUGO, S., PERALTA E., BOLUMEN, S. AND 21 SOTO-VALDEZ, H., 2005, Migration of Bisphenol A (BPA) from Can Coatings 22 into a Fatty-Food Simulant and Tuna Fish. Food Additives and Contaminants, 22(., HWEE SIAN, T., DAISHIMA, S., 2001, Determination of estrogens in 26 river water by gas chromatography-negative-ion chemical-ionization mass 27 spectrometry. J. Chromatogr. A, 919 (2), 275-282. 28 29 PARIS, F., BALAGUER, P., TEROUANNE, B., SERVANT, N., LACOSTE, C., 30 CRAVEDI, J.P., NICOLASK J.C., SULTAN, C., 2002, Phenylphenols, biphenols, 31 bisphenol-A and 4-tert-octylphenol exhibit alpha and beta estrogen activities and 32 antiandrogen activity in reporter cell lines. Mol. Cell. Endocrinol., 193, 43-49. F. AND HOGG T., 2007, Exposure assessment of chemicals from packaging 1 materials in foods: a review. Trends in Food Science & Technology, 18 (4), 219-230 2 3 RIVAS, A., OLEA, N., OLEA-SERRANO, F., 2005, Human Exposure to endocrine-4 disrupting chemicals: assesing the total estrogenic xenobiotic burden. TrAC Trends 5 Anal. Chem., 16, 613-619. 6 7 RODRIGUEZ, I., QUINTANA, J.B., CARPINTEIRO, J., CARRO, A.M., LORENZO, 8 R.A., CELA, R., 2003, Determination of acidic drugs in sewage water by gas 9 chromatography-mass spectrometry as tert.-butyldimethylsilyl derivatives. J. 10 Chromatogr. A, 985 (1-2), 265-274. 11 12 SAJIKI J., MIYAMOTO F., FUKATA H., MORI C., YONEKUBO J., HAYAKAWA K., 13 2005, Bisphenol A (BPA) and its source in foods in Japanese markets. Food Addit. 14 Contam., 24, 103-112. 15 16 SCHOENE, K., BRUCKERT, H.J., STEINHANSES, J., KONIG, A., 1994, Two stage 17 derivatization with N-(tert.-butyldimethylsilyl)-N-methyl-trifluoroacetamide 18 (MTBSTFA) and N-methyl-bis-(trifluoroacetamide) (MBTFA) for the gas-19 chromatographic analysis of OH-, SH-and NH-compounds. Fresenius J. Anal. 20 Chem., 348 (5-6), 364-370. 21 22 SHAO, B., HAN, H., HU, J., ZHAO, J., WU, G., XUE, Y., MA, Y., ZHANG, S., 2005, 23 Determination of alkylphenol and bisphenol a in beverages using liquid 24 chromatography/electrospray ionization tandem mass spectrometry. Anal. Chim. , HAN H., TU X., HUANG L., 2007, Analysis of alkylphenol and bisphenol A in

Figure 1 .

 1 FIGURE CAPTIONS

4 5Figure 2 .FoodFood

 42 Figure 2. Mass spectra of bisphenol A and surrogate (bisphenol F) in a spiked sample.

FoodTable 3 .

 3 calibration levels; a, intercept; S a , intercept standard deviation; b, slope; S b , slope standard deviation; R 2 , determination coefficient; LDR, linear dynamic range; S y/x , regression standard deviation; 05 , detection capability; P lof , P-value for lack-of-fit test.Page 23 of 24 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk Recovery assay, precision (repeatability and reproducibility) and accuracy of BPA (n = 30).

Table 1 2 3

 13 Analytical performance. Validation of the methodology

	4

Table 1 .

 1 SIM mode characterization and structural assignments of the fragments.

			Food Additives and Contaminants	Page 22 of 24
		t r (min)	Dwell time (ms)	Range (min)	Fragments (m/z)
	BPF	8.9	100	7.5 -10.0	344 [M] + , 179, 157
	BPA	10.7	100	10.0 -15.0	357 [M -Met] + , 207, 179

http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk

Table 2 .

 2 Analytical and statistical parameters of the proposed method.

	Parameter	Value
	n	6
	a	3.0•10 -3