Keith Scudamore

E Guy

Brian Kelleher

Keith ; Scudamore

Mycotoxins Kas

Robin ; Guy

Keith A Scudamore

Robin C E Guy

Susan J Macdonald

nivalenol and zearalenone during extrusion of wholemeal wheat grain

Keywords: Chromatography, Exposure assessment, Health significance Additives/Contaminants: Mycotoxins -trichothecenes, Mycotoxins -zearalenone, trichothecenes Food Types: Cereals Deoxynivalenol, nivalenol, zearalenone, Fusarium mycotoxins, wheat, flour, processing, extrusion

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Introduction

Cereals worldwide are at risk from the formation of mycotoxins both in the field as the result of infection by fusarium and other fungi or after harvest because of ineffective drying or poor storage conditions. Wheat in the field is particularly susceptible to Fusarium head blight with the likely formation of deoxynivalenol (DON), other related trichothecene mycotoxins and zearalenone (ZON). The European Commission (EC) has recently introduced maximum permissible limits for deoxynivalenol (DON) of 1250 µg/kg in raw wheat, 750 µg/kg in flour and 500 µg/kg in finished products and is currently considering legislation for other fusarium mycotoxins in order to reduce the risk to the consumer (EC, 2006).

Extrusion processing is used for producing a range of cereal products such as breakfast foods, snacks and animal feedstuffs. Commodities such as wholemeal flour, maize flour and grits are passed through the extruder under pressure, mechanical shearing stresses and elevated temperature, and expand rapidly as they are forced through the outlet die [START_REF] Riaz | Extruders in Food Applications[END_REF][START_REF] Guy | Extrusion Cooking: Technologies and Applications[END_REF]. During this process, mechanical energy is transformed into heat by viscous dissipation in the starch paste. This causes chemical bonds in the polymers to break and free radicals to form. Therefore, a contaminant such as DON is subjected both to high temperatures and to chemical reactions by free radical mechanisms and might be susceptible to some breakdown.

Temperatures inside the barrel of an extruder can be as high as 200ºC.

Extrusion processing has been shown to have variable effects on mycotoxins.

For example, approximately 85% of aflatoxins B 1 and B 2 were lost during extrusion at 150ºC when spiked corn dough was treated (Martinez and Monsalve 1989), although in a study by [START_REF] Cazzaniga | Mycotoxins inactivation by extrusion cooking of corn flour[END_REF], in which the effects of flour moisture content, temperature and the addition of sodium metabisulphite were examined, the reduction of aflatoxin B 1 was only between 10 and 25%. [START_REF] Boudra | Thermostability of ochratoxin A in wheat under two moisture conditions[END_REF] studied the decomposition of ochratoxin A under different moisture content and temperature conditions. Their results showed that ochratoxin A could be partially decomposed. In the presence of 50% water, loss is increased at 100 and 150ºC in comparison with drier grain, but is decreased at 200ºC. Some loss of ochratoxin A has been found in pilot scale extrusion, although loss below 150ºC was relatively small [START_REF] Scudamore | The fate of ochratoxin A in the processing of whole wheat grains during extrusion[END_REF]). [START_REF] Saunders | Control of fumonisin: effects of processing[END_REF] showed that extrusion of dry-milled products reduced fumonisin concentrations by 30-90% for mixing type extruders and by 20-50% for non-mixing extruders. [START_REF] Katta | Screw speed and temperature effects on stability of fumonisin B1 (FB1) in extrusion cooked grits[END_REF] examined the extrusion of corn grits on the stability of fumonisin B 1 using different temperatures and screw speeds. Both the barrel temperature and screw speed affected the loss of fumonisin in the extruded product with losses ranging from 34 to 95%. However, the loss ranged from 46-76% in those products with expansion and colour acceptable for commercial use. [START_REF] Ce | Stability of deoxynivalenol in heat-treated foods[END_REF] showed deoxynivalenol to be quite stable in heattreated foods when the effects of high temperature and high pressure processing of foods spiked with deoxynivalenol were examined. However, [START_REF] Accerbi | Utilization of highly deoxynivalenolcontaminated wheat via extrusion processing[END_REF] showed that soaking wheat naturally contaminated with high concentrations of deoxynivalenol in sodium bisulphite solution reduced levels from 7.3 to 0.8 mg kg -1 and then, after further extrusion, to 0.3 mg kg -1 .

Conflicting results in the literature may result from a failure to control or report all the conditions used. For example, chemical breakdown in the extrusion process is related to time, so that the loss of mycotoxin will depend on the residence time of the material in the extruder. The presence of minor ingredients or additives may also have a significant effect. These details are often missing from reports.

The present work forms part of a much larger study aimed at examining the fate of fusarium mycotoxins during milling and selected food processing.

Materials and methods

Wholemeal wheat containing mycotoxins

A number of wheat samples harvested from experimental plots were analysed for mycotoxins and, on the basis of the results, were mixed to provide a batch of about 200 kg of wheat with DON content of approximately 1000 µg/kg. This wheat also contained ZON at about 300 µg/kg, nivalenol (NIV) at about 200 µg/kg and lower concentrations of other related trichothecenes. The whole wheat was milled at the Campden and Chorleywood Food Association (CCFRA) using a pilot-scale Buhler milling rig as used in the Chorleywood Bread Process, but collecting all the milled fractions so as to produce wholemeal wheat flour. The Chorleywood Process was developed in 1961 by cereal scientists [START_REF] Chamberlain | The Chorleywood Bread Process[END_REF][START_REF] Axford | The Chorleywood Process[END_REF]. The flour was mixed and blended for two hours and placed in a series of polythene bags as described in a previous study on milling and baking [START_REF] Scudamore | The fate of ochratoxin A in the processing of whole wheat grains during milling and bread production[END_REF]. The moisture content of the wholemeal wheat flour produced was approximately 13%. The wheat in these bags was individually mixed again and samples of about 250 g were taken at random from each bag. The samples were then analysed separately for DON, ZON and NIV. The contents of each bag were finally recombined and re-mixed in two large batches of 65-70kg for convenience of handling during the study. After a final blending, samples from each batch were analysed again for mycotoxin content. The wholemeal wheat was calibrated for flow rate and moisture prior to setting up the processing conditions for extrusion cooking.

Processing conditions

An APV twin screw extruder model MPF 50D (supplied by APV Peterborough, UK) was set up with a screw comprised of APV type elements to form sections for conveying, mixing in water, reverse pumping in a shear zone and final high pressure pumping. The arrangement of the screw elements given in Table 1 shows a section of feed screws conveying the powder into a short mixing section of paddles and on via conveying section to the reversing paddles section. Resistance from the reversing paddles causes the section to fill and generate a pressure via the twin start screws. This in turn generates heat and melts the crystalline structures in the starch granule so that a melt fluid forms. In the final screw section the hot fluid is sealed under pressure and the water superheated to more than 140 o C and as it returns to atmospheric pressure outside the barrel the water vaporises to expand the extrudate.

The die plate contained two circular dies of 3mm diameter and 4mm land length. At the start of the extrusion cooking run the barrel heaters were set at 0/50/70/120/140 o C and adjusted to raise the barrel temperature to 160 and then 180 o C by changing the last two sections. The powder and water feed rates were adjusted to the computed settings on the plan to run at 700g/min for 15, 18 or 21% moisture w/w. The screw speed was held at 300rpm for all runs.

Experimental plan

The experimental plan involved a sequential change in extruder conditions, Table 2, commencing at 140ºC and finishing at 180ºC. The extruder was run up to a steady state at each change over and held for 2 minutes to stabilise the extrusion before a sample was collected for 2.5 minutes representing about 1.5 to 2 kg of extrudate. This was allowed to cool before packing. Logging data was taken to confirm the target settings.

Analysis

The methods of analysis used have been described previously but are summarized here.

Extraction, clean-up and determination of mycotoxins

All reagents were purchased from commercial sources and were of analytical grade.

Solvents were of HPLC grade. Mycotoxin standards of DON, NIV, and ZON were purchased from Sigma-Aldrich Company Ltd., Gillingham, Dorset, UK. DON, NIV and ZON were quantified prior to use by UV spectrometry, prepared 3-monthly.

Trichothecene charcoal/alumina clean-up columns (Tricho columns®) and zearalenone immunoaffinity clean-up columns (Zearalenone Easi-Extract®) were purchased from R-Biopharm Rhone Ltd., Glasgow.

The methods used for the trichothecenes and ZON are UKAS (ISO 17025) accredited and have performance characteristics that meet the requirements for analytical methods for mycotoxins stipulated by CEN (CEN 1999) and the EC.

Prior to analysis samples were ground to 1 mm particle size in a laboratory mill and mixed for 1 hour on either a Winkworth cone blender or Pascall laboratory scale powder mixer depending on sample size.

DON and NIV

Ground samples (20 g) were extracted with 100 ml acetonitrile/water (84:16) by shaking for 2 hours on a wrist action shaker. An aliquot of the filtered extract (5 ml) was transferred to a glass tube supplied with the charcoal/alumina clean-up column.

The plunger of the column was inserted into the tube and the column was slowly pushed through the extract at the rate of about 1 ml per 30 seconds until approximately 3 ml of cleaned up extract was collected. An aliquot (2 ml) of cleaned up extract was transferred to a 4 ml vial and taken to dryness under nitrogen using a

ZON

The method used was that of [START_REF] Macdonald | Determination of Zearalenone in Cereals and Cereal Products by Immunoaffinity Column Cleanup with Liquid Chromatography: Interlaboratory Study[END_REF]. Ground samples (25 g) were extracted with 100 ml of acetonitrile/water (75/25) by blending for 3-5 minutes and the extract was filtered. Twelve ml of the extract was diluted with 88 ml of Phosphate Buffered Saline (PBS) solution. An aliquot (50ml) of the diluted extract was passed through EASI-EXTRACT ZON immunoaffinity column (R-Biopharm Rhone) ) at a rate of no more than 3 ml/minute. The column was washed with 20 ml water and dried by passing 3 ml of air through the column. ZON was eluted at slow speed (1-2 drops per second) with 1.5 ml of acetonitrile, followed by 5 ml of air to ensure all eluate was collected An aliquot (1 ml) of the eluate was diluted with water (2 ml) and the diluted extract injected onto HPLC. These operations were carried out automatically by a Gilson ASPEc system. ZON was detected by HPLC with fluorescence detection. Liquid chromatography was performed on a either a Phenonenex ODS3-Prodgy 5 µm particle size 15 cm x 4.6 mm i.d., 250A pore size or a Spherisorb ODS2-Excel 5 µm particle size 25 cm x 4.6 mm i.d., 250A pore size operated at 1.0 ml min -1 with water : acetonitrile (45:50) as mobile phase. The excitation wavelength of the fluorescence detector was set at 275 nm and the emission wavelength was 450 nm. Sample volumes of 400 µl were injected.

Method validation and quality control

UKAS accreditation ensures that methods are valid for the tasks they are performing, but do not provide an absolute measure of accuracy. Under the Laboratory Quality System a protocol for internal and external proficiency test schemes participation is defined and the laboratory demonstrates on-going precision and accuracy. This includes analysis of in-house reference material and participation in sample testing schemes such as FAPAS (www.fapas.co.uk/fapas.cfm) and collaborative projects. Ongoing control of the methods was monitored using in-house naturally contaminated

Recovery, Limit of Detection and Determination

All analyses were conducted with a spiked sample, i.e. a known amount of toxin was added to a sample of ground wheat each day prior to extraction, clean-up and detection for each batch of samples. These samples are used to access recovery, and recoveries of 70-110% were considered acceptable. The spike level was 500 µg/kg for each trichothecene and 50 µg/kg for ZON. The limit of detection and limit of determination was 20 µg/kg and 50 µg/kg respectively for each trichothecene and 2 and 5 µg/kg for ZON. The limit of detection is defined as 3 times the electronic baseline noise and the limit of determination as the lowest level at which validation has been performed. After analysis samples were retained and stored at -20°C.

Measurement uncertainty

The analytical results have a variability known as measurement uncertainty. For any analytical method each result is reported as the best estimate for the sample and it is always qualified by a measurement uncertainty, e.g. x microgram/kg ± y microgram/kg. For this survey the calculation of the measurement uncertainty was carried out using in-house data, performance in international collaborative trials and FAPAS, thus incorporating repeatability and reproducibility data. The expanded measurement of uncertainty was calculated using a standard coverage factor of 2, equivalent to a confidence of approximately 95% that the actual level of the mycotoxin being measured lies within the quoted range. The assessment of measurement uncertainty was targeted at samples containing mycotoxins at a range of levels. The expanded measurement of uncertainty for these analyses was assessed to be ±34% at a level of 226 µg/kg ZON in wheat and ± 49% at a level of 500 µg/kg for DON and ±42% at a level of 500µg/kg for NIV.

Results from check trials

Moisture content determination

The moisture contents of the extrudates were measured by taking a 5g-sample and heating in an oven at 105ºC for 16 h.

Results and Discussion

Homogeneity of the wholemeal wheat flour

The method for testing the homogeneity of the mycotoxin concentrations in wholemeal flour was described in the method section. Table 3 gives From the 1 July 2006 values for ZON of 75 µg/kg in wheat flour and 50 µg/kg in specified consumer products have been in place within the European Union. This compares with the value around 300 µg/kg in the current flour. There are no proposals for limits for NIV but in view of its toxicology any future legislation might be at or below the limits for DON.

Analysis

The analytical methods for the analysis of DON, NIV and ZON have proven to be accurate and reliable in other studies and have been widely used for wheat and milled wheat products. In this study all recoveries fell within the accepted range of 70-110%. Sample results were corrected for the values found in spiked samples included with each analytical batch. Performance data presented here met the criteria of recovery and variability considered acceptable (CEN 1999).

Extrusion

The moisture contents of the wheat flour were 13.04±0.01 and 13.34 ± 0.02 % w/w for batches 1 and 2, respectively. It gave a linear flow relationship (R 2 ) between the speed of K-tron feeder and the weight delivered into the barrel, mass of flour g/min = 65.1 x + 97.9 where x is the speed of the K-tron.

The extrusion cooking process was run under controlled conditions for all the samples in the order shown in Table 2. Wholemeal wheat from batch 1 was used to examine the effects of experimental conditions 1-12 and from batch 2 for conditions 13-18. The running conditions were matched in all the samples for feedrate and moisture and the temperatures were set as the barrel temperatures, 140, 160 and 180 o C. The values for the feed rate during the study are also given in Table 2 and indicate that this remained quite constant throughout. This is important because if any loss of mycotoxins occurs because of chemical breakdown the time spent in the extruder becomes crucial and would introduce yet another factor if not controlled. The extruder pressure at the die depends on the conditions but increases with temperature.

The SME is a measure of the energy input to the wheat extrudate and values for specific volume indicates the degree of expansion resulting from each different condition. The largest expansion occurred at low moisture content and high temperature.

Tables 4-6 give the mean change in concentration as the % remaining for DON, NIV and ZON respectively during extrusion of wholemeal wheat flour (positive values indicate an increase in mycotoxin concentration). All results are adjusted for the moisture content of the input wheat and that of the final extrudate. Two samples were taken at each experimental set of conditions. The standard deviation between replicate pairs of extrudates is small and suggests that significant loss of DON (Table 4) occurred only in the wheat at 15% moisture content although increasing temperature did not increase loss. If DON is being lost through chemical reaction this should increase with increasing temperature. The reason for this result is unclear but could indicate that the loss is due either to binding to the wheat constituents or to poor extractability from the extruded products. At each temperature setting the loss was least in the samples extruded at a moisture content of 21%. The variability between pairs of samples was very small as shown by the low values for % SD so that these differences are real. The levels of DON were just above the maximum permissible legal limit set within the EC.

NIV is a compound structurally closely related to DON but is present here at lower concentrations which may account for rather more variation between sample pairs. At each temperature the lowest values were obtained with the lowest moisture, Table 5. However, under nearly every condition tested the concentration of nivalenol after extrusion had apparently increased. The reason for this is unclear but could be that the extrusion process is releasing 'bound' nivalenol. With 4 free hydroxyl groups NIV is more polar than DON and this might be a significant factor.

The results for ZON, Table 6 differ from those for DON and NIV in that the lowest results were in the driest samples although virtually all the ZON was recovered or slightly increased in value. ZON is much less polar than NIV or DON and this may be a factor. It is difficult to explain some of the results for these fusarium mycotoxins. However, they are all quite resistant to loss during extrusion. The apparent increase under some conditions needs further studies to confirm this. The trend for residual ZON values to tend to increase going from 140 to 180 degrees is contrary to what would be expected from chemical degradation. This is also seen for NIV but not to any extent for DON. The answer to these changes may lie with the efficiency of the extraction solvent used for removing mycotoxins in the extruded materials or that bound mycotoxins are being released by the extrusion conditions -or a combination of both.

Assessment of variability

Variability within the results can arise due to several factors. The homogeneity of the mycotoxin distribution in the wholemeal flour used, the analytical method, changes in moisture during the extrusion process, the residence time in the extruder and the efficiency of the solvent in extracting the mycotoxins from the range of extruded products formed. Data are presented for the homogeneity of the flour and the performance of the analytical method. The residence time within the extruder is One area of uncertainty remains regarding the effectiveness of extraction as all results for recovery are based on spiking. No certified reference materials exist for these mycotoxins in extruded products so this must be recognised in assessing the findings from this study. The conditions within the extruder produce a range of materials with very different physical characteristics to those of wheat flour. The products include light, highly expanded materials and hard, dense products, each physically different in structure to the wheat flour. This raises the possibility that the efficiency of solvent extraction and recovery could vary from product to product.

Conclusions

Temperature does not appear to significantly degrade any of the three mycotoxins under the extrusion conditions studied. Moisture content appears to be more important than temperature as low moisture tends to result in lower values for DON (and possibly for NIV) at all temperatures. Because loss does not appear to be affected by temperature it is probably due to binding to the wheat and/or poor extractability of the analytical solvent. Current EC legislation relies on processing to reduce DON concentrations from 750 µg/kg to 500 µg/kg and a reduction for ZON from 75 µg/kg to 50 µg/kg. This reduction appears unlikely to be achieved for some consumer products such as snacks made by a simple extrusion of wheat flour (and probably also maize flour).

  at approximately 50ºC. The residue was derivatised to form the trichothecene-trimethyl silyl (TMS) derivatives and determined by GC/MS operating in selected ion mode, using 4 ions for confirmation. Trichothecenes detected areDON, 3AcDON, 15AcDON, NIV, DAS, FUSX, T2, and HT2.

  spiked samples. Typical mean recoveries for each toxin ranged from 70 to 110 %.

  Acceptable results have been achieved in Series 22 of the FAPAS Scheme for deoxynivalenol and ZON in cereals during the period of the study (rounds 23 to 33, February 2007) as they were well inside the limits for Z scores of ± 2 considered acceptable.

  the data showing the homogeneity of the mycotoxin distribution found in the flour in samples taken at random from the two batches of wheat flour used, after the final mixing. It presents the individual and mean values for DON, NIV and ZON and the percentage standard deviation values These were considered satisfactory for the study to proceed especially as the variability of mycotoxin concentrations in the experimental samples is likely to be further minimised when the flour is mixed again in the supply hopper and during the extrusion cooking. The mean value for DON in this consignment is thus close to 1000 µg/kg. This compares with a maximum level of 750 µg/kg for DON in flour and 500 µg/kg in finished consumer products set by the EC from 1 July 2006. Thus the flour examined here should represent closely what would occur in flour containing DON at that value.

  are corrected for moisture changes. Each extruded flour sample is then compared directly with the sample of flour taken immediately before extrusion.

Table 1 .

 1 Technical detail of the arrangement of screw elements

	Screw	Length, mm
	Feed screw 1D	304.8
	Seven forwarding paddles at 45 o	88.9
	Single lead screw	152.4
	Two paddles at 90 o	25.4
	Twin start lead screw	50.8
	Two paddles 90 o	25.4
	Four reversing paddles at 45 o	50.8
	Single lead screw	50.8
	Total length	749.3

Table 2 .

 2 Sequence of extruder conditions, samples taken, and related experimental parameters at a screw rate of 300rpm and nominal feedrate of 700g/min.

								Specific
	Sample	Temperature, Feedrate, SME*, Pressure,	Moisture, %	volume
	order	o C	g/min	kJ/kg MPa Wheat flour Extrudate L/kg
	1	140	704	490	1.8	21	9.29	5.81
	2	140	703	466	1.7	21	9.48	5.73
	3	140	709	574	2.2	17	7.38	7.37
	4	140	698	571	2.0	17	7.51	7.30
	5	140	703	615	3.7	15	4.22	7.67
	6	140	682	583	3.6	15	4.61	7.52
	7	160	716	539	3.5	15	4.38	7.72
	8	160	704	537	3.5	15	4.17	7.52
	9	160	692	526	1.8	17	7.51	6.55
	10	160	675	460	1.8	17	7.74	6.61
	11	160	705	442	1.8	21	8.61	5.61
	12	160	705	441	1.6	21	8.81	5.73
	13	180	705	374	1.6	21	8.81	7.42
	14	180	705	374	1.6	21	8.95	7.25
	15	180	702	456	2.1	17	7.54	5.80
	16	180	698	466	2.1	17	7.37	5.77
	17	180	706	534	2.8	15	4.30	8.31
	18	180	706	488	2.7	15	4.05	8.38
	* SME= specific mechanical energy

Table 3 .

 3 Mycotoxin concentrations and homogeneity in wholemeal wheat flour used for extrusion study

			Mycotoxin, µg/kg
	Sample number	DON	NIV	ZON
	BATCH 1
	1a	1144	223	267
	1b	1289	179	266
	1c	1280	185	273
	1d	1368	232	280
	1e	1237	274	277
	Mean	1264	218	273
	SD	81.9	38.3	6.1
	% SD	6.5	17.5	2.2
	BATCH 2
	2a	856	198	309
	2b	1124	213	314
	2c	974	202	349
	2d	1042	198	354
	2e	848	173	360
	Mean	969	208	337
	SD	119.2	14.6	23.8
	% SD	12.3	7.4	7.1

Table 4 .

 4 Effect of extrusion on concentrations of deoxynivalenol (1264 or 969 µg/kg) corrected for moisture content changes.

	Page 17 of 19	Food Additives and Contaminants
	Extrusion Temperature, ºC	Wheat Moisture, %	Sample	DON, Surviving, %	% loss	Sample pairs, % SD
		21	a b	96.3 97.3	3.2	0.73
	140	17	a b	90.7 97.3	6.0	4.96
		15	a b	79.5 82.8	18.9	2.88
		21	a b	102.2 98.2	+0.2	2.82
	160	17	a b	96.2 104.4	+0.3	5.78
		15	a b	75.8 77.4	23.4	1.48
		21	a	101.5	0.4	1.34
			b	99.6
	180	17	a b	98.3 103.8	+1.0	3.85
		15	a b	81.9 77.2	20.5	4.18
	Mean, % SD					3.11

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Table 5 .

 5 Effect of extrusion on concentrations of nivalenol (197 µg/kg), results corrected for moisture content changes.

		Food Additives and Contaminants	Page 18 of 19
	Extrusion Temperature, ºC	Wheat Moisture, %	Sample	NIV, Surviving, %	% change	Sample pairs, % SD
		21	a b	105.1 110.8	+6.95	3.73
	140	17	a b	114.6 103.1	+8.85	7.47
		15	a b	96.5 96.4	-3.55	0.07
		21	a b	128 120.3	+24.15	4.39
	160	17	a b	110.5 113.3	+12.9	1.77
		15	a b	106.0 90.2	-1.9	11.39
		21	a	147.9	+39.9	8.09
			b	131.9
	180	17	a b	142.9 146.9	+44.9	1.95
		15	a b	132.3 132.9	+33.1	0.32
	Mean % SD					4.35

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Table 6 .

 6 Effect of extrusion on concentrations of zearalenol (305 µg/kg), results corrected for moisture content changes.

	Page 19 of 19
	Extrusion Temperature, ºC	Wheat Moisture, %	Sample	ZON, Surviving, %	% change	Sample pairs, % SD
		21	a b	86.3 79.0	-17.4	6.25
	140	17	a b	106.2 88.1	-2.9	13.17
		15	a b	113.9 116.8	+15.35	1.78
		21	a b	95.8 109.1	+2.45	9.18
	160	17	a b	110.7 101.6	+6.15	6.06
		15	a b	124.3 85.3	+4.8	26.31
		21	a	95.7	+2.45	9.32
			b	109.2
	180	17	a b	105.5 116.0	+10.2	6.70
		15	a b	104.7 110.8	+7.75	4.00
	Mean % SD					9.20

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Food Additives and Contaminants

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Acknowledgements

The work was supported by DEFRA under the Food Quality & Safety LINK programme. Wheat was supplied by Harper Adams University College, initial mixing and blending was carried by RHM Technology, High Wycombe and analysis of fusarium mycotoxins at the Central Science Laboratory, York, UK. Milling and


D B Clarke
email: db.clarke@csl.gov.uk

V Bailey

A S Lloyd

Determination of phytoestrogens in dietary supplements by LC-1 MS/MS 2

Keywords: Chromatographic analysis, Chromatography -LC/MS Additives phytoestrogens, isoflavone intake, dietary supplements, soya red clover, 24 menopause

Labeling data quantifying the exact content of individual phytoestrogen analytes in dietary 4 supplements is generally poor. As these products are commonly used in the management of 5 menopause symptoms, any clinical benefits would be dependent on the exact dosage of 6 isoflavones received. Well established extraction procedures and updated Isotope Dilution 7 Mass Spectrometry LC-MS/MS have been used to accurately quantify the concentrations of 10 8 common isoflavones in 35 dietary supplement samples on sale in the UK, Canada and Italy. 9 Concentration specific ionization suppression is described for biochanin A and formononetin. 10 All supplements contained phytoestrogens. The soya isoflavones (genistein, daidzein, 11 glycitein) were present in all products and the majority also contained the red clover 12 isoflavones (biochanin A, formononetin) and some the Kudzu isoflavones (daidzein, puerarin).

13

The content of total isoflavones per dose ranged from <1 to 53 mg. Trace amounts of 14 coumestrol were found in 6 products. Other less common analytes, the prenylnaringenins (6-15 prenylnaringenin, 8-prenylnarigenin, 6,8-diprenylnaringenin) were not found in any of the 16 products. Only 14 of 35 supplements were found to deliver more than or equal to 40 mg/day of 17 aglycone isoflavones, a consensus dose value recognized as delivering therapeutic benefit. 11 18 did not match label claims. Six delivered less than 10 mg/day isoflavones. There has been 19 little improvement in the overall quality of industry labeling in the five years since this was last 20 investigated. Consequently, the public, retailers and health care professionals should consider 21 using standardised isoflavone supplements, which are supported by analytical measurements.

The search for an alternative to HRT has lead to an ever increasing selection of 'natural' 9 alternatives becoming available in retail stores and on the internet. This raises concern, as 10 levels of the active ingredients are not always sufficient to produce the desired effect. day. As the intake of soy and isoflavones in the Asian is often cited as a possible explanation 9 of measurable health benefits (e.g. lower disease rates), clinical studies are conducted at 10 similar levels of exposure. This led to a consensus, largely by inference that dietary 11 supplements should be designed to deliver a similar intake of active ingredients i.e. 40 mg 12 day -1 isoflavones. was not conducted at the first step, as the mg quantities required in each volumetric flask were 6 impractical. Extracts were dried at 30°C under a stream of nitrogen, dissolved in 1M 7 hydrochloric acid (5 ml, aqueous) loosely stoppered and hydrolysed on a hot block at 95 0 C 8 for 2 hrs. The hydrolysed aglycones were extracted with diethyl ether (2 x 5 ml). After careful 9 separation of all aqueous acidic phase, the combined ether extracts were evaporated at 30°C 10 under a stream of nitrogen. Residues were dissolved in 1 ml HPLC mobile phase by adding 11 methanol (0.5 ml) sonicating 5 min then adding water (0.5 ml). formic acid (0.1%) and the flow ws 0.2 ml/min. The injector was programmed to provide an 2 exterior wash of acetonitrile around the needle on an extended solvent wash program (x-4).

3

The analytes quantified in samples were, genistein, daidzein, glycitein, formononetin, 4 biochanin A, coumestrol, puerarin, 6-prenylnaringenin, 8-prenylnaringenin, 6,8- For a specific phytoestrogen to be considered present in a sample extract the following criteria 10 must be met. The relative retention times of the analyte must be comparable to those of a Hydrolysis of glycosides to aglycones was confirmed by concurrent hydrolysis of labeled 5 glycoside surrogates (figure 4). Triple 13-C labeled daidzein glucoside and a set of similarly 6 labeled glucuronides of daidzein, genistein and glycitein were each hydrolysed within the 7 sample matrix. This is considered proof of quantitative hydrolysis of all glucosylated forms, to 8 their respective native aglycones using the hydrochloric acid hydrolysis protocol. This was a 9 qualitative process conducted with and without matrix, in separate vials, from the samples to 10 be quantified. normalisation of the common soya isoflavones daidzein, genistein and glycitein severely 1 limits applicability of this approach to foods that are already well characterised and known to 2 be free of formononetin (Wu 2004). The use of rarer structural isomers of more common 3 analytes, such as using prunetin an isomer of biochanin A for determination of isoflavones in 4 bovine milk makes this approach more generally applicable [START_REF] Antignac | Identification of 3 phytoestrogens in bovine milk using liquid chromatography/electrospray tandem mass 4 spectrometry[END_REF]). analogues are available and in some cases the labelled standard is being used both as a true 9 isotope dilution internal standard and as a non-labelled internal standard for a wide class of 10 analytes. This has traditionally been the only pragmatic approach for phytoestrogen analysis 11 and this has always been demonstrated to work well at low concentrations. It is often 12 overlooked that papers claiming isotope dilution generally still contain one or more analytes 13 quantified using a labelled analogue as a non-isotope dilution normalisation. A trial and error 14 approach has lead to 13 C 3 daidzein being generally accepted as the better choice than 15 13 C 3 genistein for normalisation in such cases, as it displays less intra-run variability, and 16 invariably will give data sets of lower cv%, but these will often be twice as large as those of 17 analytes quantified by true isotope dilution in the same batch. This pragmatic approach has 18 been adopted by the majority of researchers conducting LC-MS/MS measurements and there values were similar (a 1:1 ratio) and the combined total of formononetin and biochanin A was 2 about half the nominal value. At this point the quantitation method used 13 C 3 -daidzein to 3 normalised the native formononetin and biochanin A peak areas. The normalised daidzein 4 response was linear (r 2 = 0.999), while the calibration for biochanin A was non-linear, but 5 achieved an acceptable quadratic calibration (r 2 = 0.994). When only the lower concentration 6 points are used it can be seen that this deviation from linearity at higher concentrations is due 7 to the ratio of biochanin A/ 13 C 3 -daidzein changing by a factor of 2-3 fold from the predicted 8 value (figure 2). While formononetin exhibited similar properties, the deviations between 9 calculated and true values were larger for biochanin A quantitation at this stage. Using high 10 order quadratic models for fitting calibration lines is normal practice, but this approach is not 11 applicable in this example as it masks ionisation issues. As this effect appeared to be concentration based, it would be technically feasible to extract 14 differing amounts of each sample and prepare separate extracts for each analyte to measure 15 each individual analyte within a narrow dynamic range, from the LOD (0.03 µg/l) to 10 x 16 LOD (0.3 µg/ml). This would however result in a large number of chromatographic runs. 17 18 Further 13 C 3 -labelled isoflavones were obtained and this anomaly was investigated in detail. [START_REF] Booth | Confusion regarding anticoagulant coumarins in dietary 21 supplements[END_REF] While precision and between batch reproducibility were seen to be influenced by sensitivity ( 13 C 3 daidzein, 13 C 3 genistein, 13 C 3 glycitein, 13 C 3 formononetin, and 13 C 3 biochanin A) the 1 internal standard raw area response was static with the addition of up to 0.3 µg/ml of 2 unlabelled analytes (daidzein, genistein, glycitein, formononetin, biochanin A and 3 coumestrol). With increasing analyte concentration of up to 32 µg/ml, the labelled internal 4 standards display decreased ionisation. 13 C 3 biochanin A was the most reduced, with 20% 5 signal strength remaining i.e. a five-fold reduction, followed by formononetin 25%, genistein 6 38% and daidzein 40% (figure 3). If it is assumed that the unlabelled native analyte is 7 similarly suppressed, this affects the ability to accurately calibrate and calculate 8 concentrations of analytes in solution at greater than 0.3 µg/ml. This provides a very narrow 9 range in which quantitation can be achieved. In reality this effect would also be expected to 10 vary markedly between adjacent vials in an analytical run making quantitation impossible. As quantitation can be conducted by using any similar molecule as an internal standard, we 13 calculated the concentration of analyte in the reference sample (Promensil Red Clover 14 Tablets) using a number of different 13 C 3 -isoflavones. As a further comparison a higher 15 extract loading using 10-fold more extract with the same 4 µg of internal standard was 16 conducted to see how ionisations extrapolated at extreme concentrations. Without 17 concentration effects, the measured value of the concentrated solution will be 10-times that of 18 the dilute solution. For biochanin A the quantitation method using 13 C 3 -daidzein normalisation 19 was found to be the worst choice for internal standardisation and this gave 60% of the nominal 20 value at the dilute solution and 40% of nominal at the higher concentration (table 4). The 21 methoxylated derivatives of genistein and daidzein, biochanin A and formononetin were each The complete set of analyses data are listed in table 2 by individual analyte (mg/product) and 13 then as a total dose of isoflavones per dose of product and as a percentage of the labelled dose. [START_REF] Beral | Endometrial cancer and hormone-replacement 15 therapy in the Million Women Study[END_REF] The results show that there is often variability between the actual content of isoflavones 15 compared to that stated on the label. In addition, the presence of the glycoside form in many [START_REF] Boker | Intake of dietary phytoestrogens by Dutch Women[END_REF] of the soya based supplements further complicates the interpretation of the label information. 17 Based on the intake data we have described above, and an allowance of a 20% error, only 14 The red clover based products where the aglycones are extracted and solvent partitioned 1 before being added into the formulation are all deliver close to the label claim (84-133%). For 2 soya based products the accuracy of the label claim is considerably poorer (5-69%). This is 3 due to soya extracts being added by weight without specifying if conjugated to glucose or 4 being present in the free form. In the soya source material it is generally assumed that the 5 majority of isoflavones are conjugated, as an illustration, the ratio 1:52:9:38 of aglycone: 6 glucose: acetylglucose:malonyglucose will be used (Wiseman 2002). As the conjugates vary 7 in mass a matrix is used to calculate the contribution of each form to the total of aglycone 8 equivalents (table 5). Daidzein conjugates from soya as weighed out will contribute 57% 9 aglycone equivalents by mass, with genistein contributing 58% and glycitein 60%. As these 10 contributions are essentially the same irrespective of the actual ratio of daidzein : genistein : 11 glycitein in the supplement, a correction factor of 0.58 should be applied to calculate aglycone 12 equivalents. The data suggest that none of the manufacturers have applied this correction. The 13 average % of label claim for products containing soya as the sole isoflavone source average is 14 55% (excluding the outlier at 5%). After correction for aglycone equivalents against the 0.58 15 factor (58% aglycone & 42% glucose) this label claim average would have been 95%. In The three year old Novogen Red Clover tablets were found to contain identical quantities of 4 all the measured isoflavones and therefore such products are stable for three years. Where separate triplicate extracts were prepared and these also analysed in triplicate the 25 precision dropped to ± 7 %. Inter-day batch variation was in the order of ± 10 %. Tablet weights and contents of capsules varied markedly in weight. Industry figures suggest a 10% 1 variation in weight/contents is considered acceptable. Five of each tablet or capsule (contents) 2 were weighed to give an average value, which was used in the calculations. The structure of 3 the dose vehicle is also important. Capsules generally contained homogenised material that 4 can readily be weighed out without added error. Tablets contained outer coatings of e.g. iron 5 oxide that are difficult to homogenise and even when ground these coatings have a much 6 higher density, affecting homogeneity and making replicate weighings irreproducible. The 7 experimental uncertainty is therefore the sum of thes individual contributions to error and is 8 considered to be 20%, and this is due largely to sampling error not analytical performance. 9 10

Plant content of supplements 11

The listed plant ingredients on labels correlate well with the observed individual analytes. The majority of the 12 supplements were labeled as containing soya (Glycine max) and this was confirmed by analysis and the co-13 occurrence of daidzein, genistein and glycitein. A number of supplements list red clover extract (Trifolium 14 pratense) and all these products all contained elevated levels of either formononetin or biochanin A. The two 15 supplements which list Kudzu (Japanese arrowroot, Pueraria lobata) were observed to contain measurable 16 quantities of puerarin (daidzein-8-C-glucoside). As this analyte is contains a carbon-carbon bond between the 17 aglycone and sugar moieties it is resistant to the hydrolysis protocol and was measured as the intact glucoside. 18

One further product was labeled as containing "plants", as this contained puerarin, biochanin A, daidzein, 19 genistein and glycitein it probably contains a mixture of e.g. soya, red clover and kudzu. A further product was 20 labeled as containing hops. Hop flowers contain the potent phytoestrogen 8-prenylnaringenin (8-PNG) and other 21 related chemicals. As no prenyl naringenin analytes were observed in any products, it would appear that this and 22 all other products do not contain appreciable proportions of hop flowers or hop extract amongst the ingredients. 23

The public, retailers and health care professionals should therefore consider using An identical amount of internal standard gives a decreased response as the native analyte increases in concentration from vial 1 (0 ug/ml, to 0.03, 0.06, 0.16, 0.3, 0.6, 1.6, 3.2,6.4, 12.8, 16, 19 ug/ml, to vial 15 (32 ug/ml). Displayed on a concentration independent x-axis for clarity.

Introduction 1 2

 1 Vasomotor symptoms (hot flushes) are the most common menopausal symptom experienced 3 by women in Western countries. This is recognised as the main reason women seek advice 4 from their healthcare professional (Guthrie et al. 2003 Following the recent controversies 5 regarding Hormone Replacement Therapy (HRT) (Rossouw et al. 2002,Beral et al. 2005) use 6 has decreased in recent years.

11 12One

 11 group of substances receiving a great deal of interest are the phytoestrogens, in particular 13 the sub-group of compounds called the isoflavones. Phytoestrogens are naturally occurring 14 plant components that interact with mammalian endocrine systems. Evidence is accumulating 15 that isoflavones may have beneficial properties, in particular against chronic and degenerative 16 diseases including coronary heart disease and hormone-dependent cancers (Knight and Eden 17 1995, Tham et al. 1998). There is a more substantial body of evidence supporting beneficial 18 clinical effects in non-disease states such as alleviating and lowering the rates of menopausal 19 vasomotor symptoms and maintaining bone health (preventing osteoporosis) (Chiechi et al 20 2005).

21 22 Epidemiological( 16 17

 2216 data suggest that the ingestion of high isoflavone containing foods (daily 23 intake between 20 -50 mg) can result in certain health benefits. A number of clinical papers 24 and meta analyses of clinical trials using supplements containing at least 40 mg isoflavones 25 have demonstrated reduction in vasomotor symptoms (Nachtigall et al. 2006, Howes et al. are a number of other less common oestrogenic compounds such as the 1 prenylnaringenins and puerarin (daidzein-8-C-glycoside) which are also present in dietary 2 supplements. In general the aglycones exhibit much higher potency than the equivalent an extensive literature of many thousands of papers related to phytoestrogens. 6 Methods for the determination of phytoestrogens have been reviewed three times recently 7 Wang 2002, Wu 2004, Prasain 2004). The procedures for extracting, hydrolyzing and 8 chromatographically separating phytoestrogens from foods are generally well understood and 9 established, but the accepted best practice with reference to the extraction solvent, percentage 10 of water, pH and temperature, varies enormously between various food types and no single 11 ubiquitous method will be suitable to all situations (Murphy 2002, Allaouri 2005, Barbosa 12 2005). This paper uses pre-existing techniques in these areas (Grace 2003, Antignac 2003, 13 Clarke 2002, 2003a, 2003b, Wiseman 2002). The majority of phytoestrogen analyses are 14 targeted at trace level analysis, this study discusses some of the difficulties encountered when 15 conducting LC-MS measurements at high levels. Dietary intake estimates are generally based on dietary recall (DR) or food frequency 18 questionnaires (FFQ) rather than the analysis of actual diets. Both methods can be used to 19 correlate dietary intake of isoflavones directly to the consumption of primary soya products. 20 Diet analysis and DR give similar estimates where soya is deliberately consumed (Verkasalo 21 et al. 2001). Intakes of 7 and 4 mg day of genistein and daidzein in American diets have been 22 estimated from a detailed examination of 40 soya-containing products, cautioning that many 23 of the established FFQ severely underestimate isoflavone intake (Kirk et al. 1999). The European Prospective Investigation into Cancer and nutrition (EPIC study) suggests even 1 lower intake values of 0.16 and 0.15 mg day -1 each of genistein and daidzein (Boker et al. 2 2002). Dietary intake of isoflavones in Asian soya diets has been estimated on numerous 3 occasions and is now generally accepted to be in the range of 30-50 mg day -1 of the combined 4 isoflavone aglycone equivalents (daidzein, genistein and glycitein) (Wakai et al. 1999, 5 Messina 1995). The latest estimate (Kikuchi et al. 2001), a market basket approach of broadly 6 similar design to the UK Total Diet Study (Peattie et al. 1983), analysed Japanese diets for the 7 hydrolysed aglycones. This provided an intake figure for combined aglycones of 38 mg per 8

13 14 The4

 14 two previous works in this area that used branded products highlighted problems in the 15 isoflavone supplement industry (Howes and Howes 2002, Setchell et al. 2001). Firstly many 16 manufacturers used the weight of glucosides as the measure of potency rather than adjusting 17 downwards for the mass of aglycone. Subsequently many products claimed to deliver an 18 appropriate quantity of the active principle. As an example, in the Howes letter, a label claim 19 of 68 mg isoflavones/dose was supported by an analytical measurement of 50 mg, but this 20 drops to 31 mg when corrected for aglycone content. This product and others like it then 21 deliver less than the stated dose. In that study, only one of the nine products, on sale in 22 Australia, delivered the stated dose measured (38 mg label 40 mg measured), the other eight 23 were found to deliver less than half the stated dose (Howes and Howes 2002). In the other 24 larger study of 33 American dietary supplement products, Setchell measured the total 25 isoflavone weight, including glucosides and found that half deviated by more than 10% from 26 values. Six products were more than 50% from the claimed value. Eight of the 33 1 products delivered 40-60 mg/day total isoflavones. The majority therefore did not deliver 40 2 mg aglycones (Setchell et al. 2001) 3 This study has measured the concentrations of isoflavones in a number of isoflavone 5 supplements available in the UK, Canada and Italy. In addition, we have looked at the 6 presence of any other substances that may have an adverse impact on health or interact with 7 pharmaceutical therapies. Previous studies conducted in USA and Australia, showed that there 8 is a great variability in composition of isoflavone supplements, as determined by High 9 Performance Liquid Chromatography (HPLC-UV) profiles, many containing peaks of 10 unknown origin and chemical structure and that for over half the products tested the actual 11 isoflavone levels deviated by more than 10% from the claimed value (Setchell et al. 2001, 12 Howes and Howes 2002).

  intact retail packaging were collected by the authors or donated by 20 Novogen Ltd. Samples of red clover, red clover extracts, test certificates and an analysis 21 protocol were supplied by Novogen Ltd. Novogen Red Clover Sample 35 was a older version 22 of sample 14, this three year old formulation sample was assayed as a quality control check to 23 confirm long term product stability at room temperature in the final packaging. Tablets were 24 weighed intact. Capsules were weighed full, then opened and the contents emptied out and the 25 empty capsules and contents weighed separately. All products were weighed in batches of five 26 and the average weights used. All samples were pulverised to powder prior to analysis and 27 stored at ambient conditions. solvents and chemicals were of analytical grade or equivalent. The 2 phytoestrogens; daidzein, genistein, glycitein, biochanin A, formononetin and coumestrol 3 were purchased from Apin Chemicals (Abingdon, UK). [2,3,4-13 C 3 ]formononetin, [2,3,4-4 13 C 3 ]biochanin A, [2,3,4-13 C 3 ]daidzein, [2,3,4-13 C 3 ]daidzein glucoside, [2,3,4-13 C 3 ]daidzein 5 glucuronide, [2,3,4-13 C 3 ]genistein, [2,3,4-13 C 3 ]genistein glucuronide, [2,3,4-13 C 3 ]glycitein 6 and [2,3,4-13 C 3 ]glycitein glucuronide were supplied by Dr Nigel Botting (St. Andrews 7 University, UK). Puerarin, 6-prenylnarinegnin, 8-prenylnaringenin, 6,8-diprenylnaringenin 8 were purchased form Plantech (Reading UK). The reference compounds were stored as 9 powders at -18 o C. Stock solutions were prepared in ethanol, or ethanol/water (polar 10 conjugates) and stored at +4 o C. The purity of standards was assessed by HPLC-UV, any 11 containing observable quantities of contaminants or by products were discarded. The purity of 12 the bulk stock of daidzein and genistein analytical standards was further assessed at the 13 beginning of this work against values from by an inter-laboratory trial where five European 14 laboratories provided extinction coefficient data on separately sourced standards (Wiseman et 15 al. high concentrations of isoflavones, and these being presented as pre-extracted and 19 semi-purified active principles, often as the aglycone, these products achieve optimum 20 solubility in a different solvent mix to that used to extract intact soya products. An industry 21 protocol was used to solubilise products. Once solubilised the analysis scheme of acid 22 hydrolysis and Isotope Dilution Mass Spectrometry was similar to that used in previous 23 investigations into phytoestrogen content of food (Clarke et al. 2003a and 2003b).

  0.080 ± 0.001g) were weighed into silyanised glass vials and were 27 quantitatively transferred with solvent into volumetric flasks by the following procedure. Add 28 ethanol (3.5 ml) and sonicate in vial for 5 min, transfer to 50 ml volumetric flasks. Add a 29 ethanol water mixture (40 ml, 1:1), in portions to quantitatively transfer all extract and solid 30 volumetric flask, which was sonicated a further 30 mins. After the solution 1 had cooled, the extracts were made up to the 50 ml mark with further ethanol water. A portion 2 (0.200 ml) of each extract was dispensed into a silyanised glass vial and a solution of internal 3 standard was added (100 µl containing [2,3,4-13 C 3 ]daidzein, [2,3,4-13 C 3 ]glycitein, [2,3,4-4 13 C 3 ]formononetin [2,3,4-13 C 3 ]biochanin A and [2,3,4-13 C 3 ]genistein). Internal standardization 5

5 diprenylnaringenin.

 5 Structures are summarized in Figure 1. Analytes were quantified in the 6 range 0.03 to 48 µg/ml.

  9

11 retention

 11 time marker,(the first eluting internal standard), the internal standard (used for 12 analyte quantitation i.e. native and 13 C 3 -analogue are within ± 0.05 min), and to authentic 13 analytical standards of each analyte. The peak must have the correct mass transition, 14 maximizing at the correct retention time. The signal to noise ratio of any peak must be greater 15 than 3:1. All samples were analysed from duplicate weighings with single analytical runs for 16 each extract, mean values of these duplicate analyses were used. Each analytical batch 17 contained reagent blanks, spiked reagent blanks, replicate analyses and reference samples. As 18 an example, in the definitive analytical batch, the Novogen RedClover and Promensil 19 reference material were each analysed from three separate crushed tablets, with duplicate 20 weighings from each of the three powdered tablets (3 x 2 = 6 replicates). 21 Samples of known composition were used to validate the analytical approach. A number of 22 raw materials, extracts and formulated products were supplied with the manufacturers 23 analytical test certificates. This was further assessed by an initial comparison to an HPLC-UV 24 method supplied by Novogen Ltd. The two methods were judged as comparable when 25 within 10% of nominal values. The Novogen product Promensil was used to 1 assess method variability by replicate extractions and analyses in the final experiment.

Food

  the isoflavone genistein, daidzein, glycitein, formononetin, 16 biochanin A, were observed in many of the products. Puerarin was observed as a minor 17 component in three products. No products contained significant levels of coumestrol. Many 18 products which are labelled as containing phytoestrogen ingredients contained lower 19 quantities of isoflavones than expected (table3). continue to successfully use non-labelled and chemically dissimilar, 26 molecules as the internal standard, such as flavone for measuring isoflavones in vegetables 27[START_REF] Antonelli | Simultaneous 9 quantitation of free and conjugated phytoestrogens in Leguminosae by liquid 10 chromatography-tandem mass spectrometry[END_REF], The simpler design of using formononetin as internal standard for Page 10 of 38 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk

5 6

 5 While isotope dilution is a powerful technique, it has many of the same limitations as other 7 internal standardisation techniques, especially when only a limited number of labelled 8

19 have

 19 been few problems reported for low level quantitation in matrices such as serum and 20 urine (Grace 2003, Clarke 2002).21 22 It was noticed that the two principle red clover analytes, formononetin and biochanin A were 23 failing to give adequate values for a reference material in initial range finding experiments. 24 Further investigation by comparison of UV responses from running the industry HPLC-UV 25 analysis method lead us to believe that the supplied values were correct and that the ratio of 26 formononetin in the reference sample was ca. 1.6. By LC-MS the calculated 1

  20 and instrument cleanliness, self suppression was observed to correlate with increasing native 21 analyte concentration in the calibration solutions. This was a factor of total analyte load at 22 each point in the charomatogram, andnot of the individual concentrations. The point where 23 self-suppression was induced therefore varied batch by batch and occurred at a lower 24 concentration of each individual analyte as labelled internal standard were introduced, 25 increasing the analyte loading. In solutions containing 4 µg/ml of the internal standards 26

  11 12

  22better individual internal standard choices for the normalisation of both biochanin A and 23 formononetin than either of the parent hydroxyl analytes genistein and daidzein as would be what we report is academically very obvious, such fundamental ionisation and 1 self-suppression issues resulting in an inability to use chemically dissimilar internals standards 2 has not been previously been reported and we believe these problems are due primarily to 3 operating at the elevated concentrations required for dietary supplements analysis.

4 5

 4 In summary, when the method was extended to include further internal standards, one for each 6 of the major analytes being measured the errors introduced through self-suppression were 7 overcome allowing accurate quantitation over a further two orders of magnitude of

  18 out of 35 supplements had sufficient levels, i.e. a minimum of 40 mg of aglycone isoflavones 19 to achieve the intended therapeutic benefit. When compared to label claims, only 11 of 35 20 were within ± 20% of the stated value. There was significant overlap, whereby 8 of the 14 21 products that contained 40 mg/day of aglycone isoflavones were also within ± 20% of the 22 stated value. 6 products were found to contain less than 50% of the stated dose. Our study 23 shows that there has still been no overall improvement in labelling quality from previous 24 studies (Howes et al. 2002, Setchell et al. 2001).

  16summary, manufacturers of soya supplements are putting in the correct weight, but of the 17 wrong form of isoflavone and these products do not deliver the stated dose. For mixed soya ; 18 red clover or soya kudzu it appears some are correctly formulated (ca. 100% of label claim) 19 and some are not (ca. 40-60% label claim) 20 21 Either soya or soya germ, the hypocotyl portion of the soy bean is used as high concentration 22 starting material Our previous work has shown that soya germ can provide a three fold 23 increase in isoflavone content relative to the whole bean. The ratios of the three isoflavone 24 daidzein, genistein and glycitein changes from 7:10:1 in soya flour to 4:1:3 in soya germ. 25 Changing from a high genistein to high daidzein/glycitein product affects the potency as the 26 oestrogenic potency. The two different profiles, of high genistein, or high 1 daidzein/glycitein were readily observed in these supplements.2 3

7

  concern and confusion between coumestans such as coumestrol, a 8 benzofurocoumarin and coumarins such as coumadin (warfarin), which exhibit 9 antithrombotic-anticoagulant effects. Most naturally occurring coumarins have not been 10 evaluated, but some can be harmful. Dicoumarol was found to be the compound responsible 11 for hemorrhagic death of livestock consuming mouldy hay. Standardized red clover has been 12 clinically evaluated and the extract presents little risk of coagulation abnormalities (Booth et 13 al. 2004). A dose of 25 to 200 mg/day of dicoumarol is needed to produce clinically 14 significant anticoagulation, this level of intake is virtually impossible to achieve by ingestion 15 of 500 mg dietary supplement tablets. Supplement manufacturers generally consider it 16 cautionary to ensure that no coumestrol is present. None of the products studied contained 17 high levels of coumestrol. the glycosides with concomitant appearance of labelled aglycone is 22 taken as proof of successful hydrolysis of native analytes (figure 4). Daidzein glucoside was 23 the only labelled glucoside currently available, therefore a larger set of isoflavone 24 glucuronides surrogates (daidzein, genistein, glycitein) were used alongside daidzein 25 glucoside to probe genistein and glycitein hydrolysis. In the first pair of chromatograms the to contain the labelled glucoside conjugate (MRM 432>256) and to be clear 1 of the labelled aglycone (MRM 256>226), while after hydrolysis, the conjugate is removed 2 and the aglycone is observed. In the same hydrolysed sample with and without addition of the 3 labelled daidzein glucuronide, the native un-labelled daidzein (MRM 253>223) is observed to 4 be unchanged in concentration while the labelled glucuronide is hydrolysed. Similar profiles 5 are obtained for the daidzein glucoside, the genistein and glycitein glucuronides 6 For this experiment it was first necessary to update our previous instrumental method to use 8 the same labelled aglycones for the core isotope dilution mass spectrometry quantitation 9 (IDMS) as are released from the hydrolysed conjugates. Due to ongoing improvements in 10 synthesis, the set of available aglycone, glucoside and glucuronide standards available are all 11 now labelled in identical positions [2,3,4-13 C 3 ], with the three labelled atoms adjacent to each 12 other in the central ring. It was essential to examine the fragmentation patterns of the 13 aglycones and to establish if the ring cleavage would produce different mass fragments for 14 standards with different labelling patterns. In the two examples synthesised to date our 15 previous internal standard [3,4,1'-13 C 3 ]-genistein and the new [2,3,4-13 C 3 ] fortuitously 16 yielded the same mass fragment as the predominate ion (figure 5). It is therefore not necessary 17 to make any instrumental alterations in order to utilise the benefit of these new standards. 18 Care must be taken as fragmentation of other labelling such as [3,4,8-13 C 3] ]-genistein will 19 result in a need to monitor a different mass fragment. 20 21 Precision of the method and experimental uncertainty was due to contributions from four 22 separates processes. Analytical precision was calculated by conducting replicate analyses of 23 sample extracts. With either three or six replicates the instrumental precision was within ± 2%.

24

 24

24 standardisedFood 12 Phytomedicine, 14 , 24 Page

 24121424 isoflavone supplements, which are supported by analytical measurements and the 25 content of active components adjusted accordingly. of 35 dietary supplements intended for treatment of menopausal 30 symptoms have been measured. 14 out of 35 contained concentrations of aglycone isoflavones 31 that provide a comparable intake with a high soya diet and are within the accepted therapeutic 32 range for natural relief of menopausal symptoms by the intake of isoflavones. 11 out of the 35 33 Page 18 of 38 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk not match label claims and six delivered less than 10 mg/day of 1 isoflavones. This study highlights the need for careful selection of isoflavone containing liquid chromatography-tandem mass spectrometry with [ 13 C 3 ]-1 isoflavone internal standards. Analytical Biochemistry, 309(1), 158-172.Clarke, D. B, 2 Barnes, K. A. and Lloyd, A. S., 2003, Determination of unusual soya and non-soya 3 phytoestrogen sources in beer, fish products and other foods. Food Additives and 4 Contaminants, 21(10), 949-962. 5 Clarke, D. B, and Lloyd, A. S., 2003, Dietary exposure estimates of isoflavones from the 1998 6 UK Total Diet Study. Food Additives and Contaminants, 21(4), 305-316. 7 de Kleijn, M. J. J., Van der Schouw, Y. T., Wilson, P. W. F., Adlercreutz, H., Mazur, W., 8 Grobbee, D. E., & Jacques, P. F., 2001,. Intake of dietary phytoestrogens is low in 9 postmenopausal women in the United States: The Framingham Study. Journal of Nutrition, 10 131, 1826-1832. 11 Geller, S. E., and Studee, L. Botanical and dietary supplements for menopauseal symptoms: 12 what works, what doesn't. J. Womens Health (Larchmt). 2005 14(7), 634-649. 13 Grace P. B., Taylor, J. I., Botting, N. P., Fryatt, T., Oldfield, M. F., Al-Maharik, N., % 14 Bingham, S. A. 2003, Quantitation of isoflavones and lignans in serum using isotope 15 dilution liquid chromatography/tandem mass spectrometry. Rapid communications in mass 16 spectrometry, 17(12), 1350-1357Guthrie, J. R., Dennerstein, L., Taffe, J. R., and 17 Donnelly, V., 2003, Health care-seeking for menopausal problems, Climacteric, 6(2), Isoflavones in healthy humans and analysis of commercial soy isoflavone supplements. 1 The Journal of Nutrition, 131(4 suppl), 1362S-75S. 2 Setchell, K. D. R., Brown, N. M. Zimmer-Nechemias, L., Brashear, W. T., 2002, Evidence for 3 lack of absorption of soy isoflavone glycosides in humans, supporting the crucial role of 4 intestinal metabolism for bioavailability. American Journal of Clinical Nutrition, 76(. M., Gardner, C. .D, and Haskell, W. L. 1998, Clinical review 97: Potential Health 7 Benefits of Dietary Phytoestrogens: A review of the clinical, epidemiological and 8 mechanistic evidence. Journal of Clinical Endocrinology and Metabolism, 83 (7), 2223-9 35. 10 Thompson Coon, J., Pittler, M. H., Ernst, E. 2007, Trifolium pratense isoflavones in the 11 treatment of menopausal hot flushes: A systematic review and meta-analysis. 153-159. 13 Verkasalo, P. K., Appleby, P. N., Allen, N. E., Davey, G., Adlercreutz. H., & Key, T. J., 2001, 14 Soya intake and plasma concentrations of daidzein and genistein: validity of dietary 15 assessment among eighty British women (Oxford arm of the European Prospective 16 Investigation into Cancer and Nutrition). British Journal of Nutrition, 86, 415-421. 17 Wakai, K., Egami, I., Kato, K., Kawamura, T., Tamakoshi, A., Lin, Y., Nakayama, T., Wada, 18 M., & Ohno, Y., 1999, Dietary intake and sources of isoflavones among Japanese. 19 Nutrition and Cancer, 33, 139-145. 20 Wiseman, H., Casey, K., Clarke, D. B., Barnes, K, & Bowey, E, 2002, The isoflavone 21 aglycone and gluco-conjugate content of commercial and "home-prepared" high soy and 22 low soy foods and food dishes in the UK selected for use in chronic controlled nutritional 23 studies. Journal of Agricultural and Food Chemistry, 50, 1404-1419.

Figure 1 .Figure 2 .Figure 3 .

 123 Figure 1. Common dietary sources and chemical structures of phytoestrogens contained in dietary supplements

  Com pound nam e: D aidzein Coefficient of D eterm ination: R^2 = 0.999290 Calibration curve: 0.000125223 * x^2 + 0.0792306 * x + 0.000445503 Res pons e type: Internal Std ( Ref 1 ), Area * ( IS C onc. / IS Area ) Curve type: 2nd Order, Origin: Exclude, Weighting: 1/x, Axis trans : e: Biochanin A Coefficient of D eterm ination: R^2 = 0.988121 Calibration curve: 7.66061 * x Res pons e type: Internal Std ( Ref 1 ), Area * ( IS C onc. / IS Area ) Curve type: Linear, Origin: Force, Weighting: 1/x, Axis trans : e: Biochanin A Coefficient of D eterm ination: R^2 = 0.994452 Calibration curve: -0.0368557 * x^2 + 2.87369 * x + 1.28066 Res pons e type: Internal Std ( Ref 1 ), Area * ( IS C onc. / IS Area ) Curve type: 2nd Order, Origin: Include, Weighting: Null, Axis trans :

Figure 1 .Figure 2 .Figure 3 .

 123 Figure 1. Common dietary sources and chemical structures of phytoestrogens contained in dietary supplements

Figure 5 .

 5 Figure 5. LC-MS/MS fragmentation of labelled standards. Identification; A) [2,3,4-13 C 3 ]Daidzein, B) [3,4,8-13 C 3 ]Daidzein. For labelled daidzein, as the MRM transitions are a simple doubledehydroxylation the position of labelling is unimportant. For labelled genistein the MRM fragment is derived from a ring cleavage and the positioning of label is important. In the two examples synthesised to date C) [2,3,4-13 C 3 ] and D) [3,4,1'-13 C 3 ]genistein, fortuitously chemically dissimilar fragments of the identical mass fragment are obtained. Fragmentation of other patterns such as [3,4,8-13 C 3] ] genistein will result in a need to monitor a different mass fragment.

Data were acquired by tandem mass spectrometry using a multiple reaction monitoring 17 (MRM) method that monitored parent to daughter ion transitions for each analyte. Individual 18 transitions, retention times and ionization parameters are listed in Table 2. The desolvation

	1	at 26 min and a 4 min equilibration before the next cycle. Both mobile phases contained
		F o r
		P
		e
	12	e r
	13 14 15 16 UK). 19 Chromatography and quantitation R Analyses were conducted on a Micromass Ultima triple quadrupole mass spectrometer e equipped with an electrospray source operating in negative ion mode (Waters, Manchester, v i e w temperature was 300 o C and the source block temperature was 100 o C. The desolvation gas 20 O n l
	21	flow was 700 L/h, the cone gas flow was 100 L/h and the capillary voltage was 2.8 Kv. y
	22	Reversed phase separations were carried out at 30 o C using a Waters Bridged Ethylene Hybrid
	23	(BEH) C18 column (2.1 x 150 mm) (Waters, Manchester, UK). The injection volume was
	24	generally 20 µl. A gradient program was used, starting from 10% acetonitrile with 90%
	25	aqueous, holding for 3 mins and changing to 30% acetonitrile at 7 min, increasing to 100%
	26	acetonitrile at 23 min, with a 2 min hold at 100% acetonitrile and a return to 10% acetonitrile
		9

manuscriptcentral.com/tfac Email: fac@tandf.co.uk Food Additives and Contaminants

	1
	F o r F o r
	P P
	e e
	e r e r
	R R
	e e
	v i e v i e
	w w
	O n l O n l
	y y

Table 1 .

 1 Isoflavone parent aglycones and glucosides

	2
	Aglycone	Glycoside
	Genistein	Genistin
	Daidzein	Daidzin
	Glycitein	Glycitin
	Biochanin A	Sissotrin
	Formononetin	Ononin
	3

Table 2 .

 2 Acquisition parameters for the quantitation of phytoestrogens

	Page 25 of 38
	4
	5
	Compounds	Retention	MRM	Dwell	Collision	Cone
		time	transition	time	Energy	Voltage
		(mins)	(m/z)	(ms)	(eV)	(V)
	Puerarin	4.7	415→295	0.05	50	30
	Daidzein	14.0	253→223	0.05	60	25
	13 C 3 -Daidzein	14.0	256→226	0.05	60	25
	Glycitein	14.4	283→268	0.05	50	30
	13 C 3 -Glycitein	14.4	286→271	0.05	50	20
	Genistein	15.6	269→133	0.05	55	30
	13 C 3 -Genistein	15.6	272→135	0.05	55	30
	Coumestrol	15.7	267→266	0.05	60	27
	Formononetin	17.1	267→252	0.05	60	23
	13 C 3 -Formononetin	17.1	270→255	0.05	40	22
	Biochanin A	18.9	283→268	0.05	50	20
	13 C 3 -Biochanin A	18.9	286→271	0.05	50	20
	6-Prenylnaringenin	19.0	339→219	0.05	50	20
	8-Prenylnaringenin	20.8	339→219	0.05	50	20
	6,8-Diprenylnaringenin	23.7	407→119	0.05	50	20
	6
	7
	8
	9

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n l y 26Table 3 .

 3 Measured content of phytoestrogens in dietary supplements: daily doses and % of nominal content.

			Weight		Individual analytes mg/unit	*	Total isoflavones	Units/ Actual dose	Label claim	% of
	Product type		g/unit	Gen Daid Gly Form Bio Cou Pur	mg/unit	Day #	mg/day	mg/day †	Label claim
	Soya	Tab	1.06		0.2	0.7	0.2	0.0 0.1 0.00 0.00	1	1	1	2.2	45
		Caps	0.34		1.3	6.7	2.1	0.0 0.0 0.00 0.00	10	2	20	35	57
		Caps	0.45		0.3	0.3	0.0	0.0 0.0 0.00 0.00	1	2	2	n/a	-
		Caps	0.54		13.6 10.8 2.4	0.0 0.0 0.01 0.00	27	1	27	40	68
		Tab	1.17		10.3 11.0 1.6	0.0 0.0 0.00 0.00	23	1	23	40	58
		Tab	0.65		1.9 11.7 4.3	0.0 0.0 0.00 0.00	18	1	18	38	47
		Caps	0.31		8.1	4.1	0.2	0.0 0.0 0.00 0.00	12	1	12	60	20
		Tab	1.13		15.6 10.7 1.5	0.0 0.0 0.00 0.00	28	1	28	50	56
		Caps	0.55		0.8	2.3	2.3	0.0 0.0 0.00 0.00	5	3	16	37.5	43
		Tab	1.09		12.6 12.9 2.0	0.0 0.0 0.00 0.00	28	1	28	50	56
		Caps	1.01		0.3	0.9	0.3	0.0 0.0 0.00 0.00	1	1	1	20	5
	F o r	Tab Caps Tab Tab Tab Tab Caps	1.10 0.77 0.88 0.69 0.68 0.70 0.46		14.0 12.0 2.5 1.7 7.9 3.0 14.6 21.1 4.7 15.5 8.5 0.5 16.7 8.8 0.5 19.3 17.4 2.1 5.3 5.1 0.8	0.0 0.0 0.00 0.00 0.0 0.0 0.00 0.00 0.0 0.0 0.00 0.00 0.0 0.0 0.00 0.00 0.0 0.0 0.00 0.00 0.0 0.0 0.01 0.00 0.0 0.0 0.00 0.00	29 13 40 25 26 39 11	1 2 1 1 1 1 2	29 25 40 25 26 39 23	55 46 60 37.5 37.5 60 40	53 54 67 67 69 65 58
	Caps Caps Tab P Caps Novogen -Redclover exp 2008 Redclover Tab e Novogen-RedClover exp 2005 Tab Novogen -Promensil Tab Caps e 0.58 0.47 0.80 0.56 0.48 0.54 0.53 0.71 r	11.2 10.6 1.5 0.6 0.2 0.4 18.9 16.6 0.00 0.00 0.0 0.0 0.00 0.00 1.6 4.7 5.2 30.2 7.9 0.02 0.00 1.9 2.9 2.6 24.6 5.5 0.01 0.00 1.0 0.5 0.6 15.8 23.6 0.00 0.00 0.8 0.5 0.5 15.2 23.7 0.00 0.00 1.0 0.5 0.6 17.1 24.9 0.00 0.00 1.4 2.6 2.4 36.5 10.5 0.01 0.00	23 37 50 37 41 41 44 53	1 1 1 1 1 1 1 1	23 37 50 37 41 41 44 53	40 40 40 44 40 40 40 40	58 93 125 84 103 103 110 133
		Tab Tab Caps	0.54 0.77 0.47 R 1.1 0.3 0.6	0.4 1.4 0.1	0.4 16.9 25.6 0.00 0.00 1.9 17.7 4.8 0.00 0.00 0.2 24.0 18.6 0.00 0.00	44 26 43	1 2 1	44 52 43	40 40 40	110 130 108
	Soya and Redclover Kudzu Hops	Caps Caps Caps Caps Tab Tab Tab Caps	0.35 0.72 0.72 0.40 0.55 1.05 1.62 0.31	3.3 23.1 7.7 7.5 11.7 0.6 e 8.1 12.7 0.6 0.9 2.4 0.6 2.7 6.4 5.8 3.9 8.8 8.9 1.7 2.8 2.5 31.1 8.3 0.01 0.02 1.2 0.3 0.00 0.00 0.7 0.5 0.00 0.00 0.7 0.5 0.00 0.00 1.2 0.3 0.00 0.00 0.9 0.5 0.04 0.24 0.2 0.1 0.05 0.14 v i e 1.3 6.1 2.0 0.0 0.0 0.00 0.00 w	36 21 22 6 16 22 46 9	1 1 1 2-3 1 1 1 1-2	36 21 22 11 16 22 46 19	62.5 20 20 20 35 55 40 17.2	58 105 110 55 46 40 115 110
							O n l
								y
					27

*

Gen = genistein, Daid = daidzein, Gly = glycitein, Form = formononetin, Bio = biochanin A, Cou = coumestrol, Pur = puerarin. # Where a dose range is given e.g. 2-3 per day the lower value has been used.

†

The dose is assumed to be measured as the aglycones.

Table 4 .

 4 Differences in quantitation of biochanin A and formononetin in Promensil

	Analyte	Internal std		µg/ml
			Diluted Concentrrated
	Biochanin A	13 C 3 -Genistein	10.6	54
		13 C 3 -Daidzein	7.2	32
	13 C 3 -Biochanin A	12.8	131	Correct value
	13 C 3 -Formononetin	10.3	90
	Formononetin	13 C 3 -Genistein	8.9	48
		13 C 3 -Daidzein	6.1	31
	13 C 3 -Biochanin A	8.7	78
	13	C 3 -Formononetin	10.8	103	Correct value

Table 5 .

 5 Calculation of aglycone equivalents in soya based products

			Molecular	Aglycone	Composition	Aglycone
	Compound		weight	equivalents	ratio	equivalents
	Daidzein	Aglycone	254	1.00	0.01	0.01
		Daidzin	416	0.61	0.52	0.32
		6''-O-Acetyldaidzin	458	0.55	0.09	0.05
		6''-O-Malonyldaidzin	502	0.51	0.38	0.19
					1.00	0.57
	Genistein	Aglycone	270	1.00	0.01	0.01
		Genistin	432	0.63	0.52	0.33
		6''-O-Acetylgenistin	479	0.56	0.09	0.05
		6''-O-Malonylgenistin	518	0.52	0.38	0.20
					1.00	0.58
	Glycitein	Aglycone	284	1.00	0.01	0.01
		Glycitin	446	0.64	0.52	0.33
		6''-O-Acetylglycitin	488	0.58	0.09	0.05
		6''-O-Malonylglycitin	532	0.53	0.38	0.20
					1.00	0.60
			Daidzein/Genistein/Glycitein ratio of 4:1:3	0.58
			Daidzein/Genistein/Glycitein ratio of 7:10:1	0.58

Table 1 .

 1 Isoflavone parent aglycones and glucosides

	2
	Aglycone	Glycoside
	Genistein	Genistin
	Daidzein	Daidzin
	Glycitein	Glycitin
	Biochanin A	Sissotrin
	Formononetin	Ononin
	3
	4
	5

Table 2 .

 2 Acquisition parameters for the quantitation of phytoestrogens

	6
	7
	Compounds	Retention	MRM	Dwell	Collision	Cone
		time	transition	time	Energy	Voltage
		(mins)	(m/z)	(ms)	(eV)	(V)
	Puerarin	4.7	415→295	0.05	50	30
	Daidzein	14.0	253→223	0.05	60	25
	13 C 3 -Daidzein	14.0	256→226	0.05	60	25
	Glycitein	14.4	283→268	0.05	50	30
	13 C 3 -Glycitein	14.4	286→271	0.05	50	20
	Genistein	15.6	269→133	0.05	55	30
	13 C 3 -Genistein	15.6	272→135	0.05	55	30
	Coumestrol	15.7	267→266	0.05	60	27
	Formononetin	17.1	267→252	0.05	60	23
	13 C 3 -Formononetin	17.1	270→255	0.05	40	22
	Biochanin A	18.9	283→268	0.05	50	20
	13 C 3 -Biochanin A	18.9	286→271	0.05	50	20
	6-Prenylnaringenin	19.0	339→219	0.05	50	20
	8-Prenylnaringenin	20.8	339→219	0.05	50	20
	6,8-Diprenylnaringenin	23.7	407→119	0.05	50	20

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Table 3 .

 3 Measured content of phytoestrogens in dietary supplements: daily doses and % of nominal content.

			Weight		Individual analytes mg/unit	*	Total isoflavones	Units/ Actual dose	Label claim	% of
	Product type		g/unit	Gen Daid Gly Form Bio Cou Pur	mg/unit	Day #	mg/day	mg/day †	Label claim
	Soya	Tab	1.06	0.2	0.7	0.2	0.0 0.1 0.00 0.00	1	1	1	2.2	45
		Caps	0.34	1.3	6.7	2.1	0.0 0.0 0.00 0.00	10	2	20	35	57
		Caps	0.45	0.3	0.3	0.0	0.0 0.0 0.00 0.00	1	2	2	n/a	-
		Caps	0.54	13.6 10.8 2.4	0.0 0.0 0.01 0.00	27	1	27	40	68
		Tab	1.17	10.3 11.0 1.6	0.0 0.0 0.00 0.00	23	1	23	40	58
		Tab	0.65	1.9 11.7 4.3	0.0 0.0 0.00 0.00	18	1	18	38	47
		Caps	0.31	8.1	4.1	0.2	0.0 0.0 0.00 0.00	12	1	12	60	20
		Tab	1.13	15.6 10.7 1.5	0.0 0.0 0.00 0.00	28	1	28	50	56
		Caps	0.55	0.8	2.3	2.3	0.0 0.0 0.00 0.00	5	3	16	37.5	43
		Tab	1.09	12.6 12.9 2.0	0.0 0.0 0.00 0.00	28	1	28	50	56
		Caps	1.01	0.3	0.9	0.3	0.0 0.0 0.00 0.00	1	1	1	20	5
		Tab	1.10	14.0 12.0 2.5	0.0 0.0 0.00 0.00	29	1	29	55	53
		Caps	0.77	1.7	7.9	3.0	0.0 0.0 0.00 0.00	13	2	25	46	54
		Tab	0.88	14.6 21.1 4.7	0.0 0.0 0.00 0.00	40	1	40	60	67
		Tab	0.69	15.5 8.5	0.5	0.0 0.0 0.00 0.00	25	1	25	37.5	67
		Tab	0.68	16.7 8.8	0.5	0.0 0.0 0.00 0.00	26	1	26	37.5	69
		Tab	0.70	19.3 17.4 2.1	0.0 0.0 0.01 0.00	39	1	39	60	65
		Caps	0.46	5.3	5.1	0.8	0.0 0.0 0.00 0.00	11	2	23	40	58
		Caps	0.58	11.2 10.6 1.5	0.0 0.0 0.00 0.00	23	1	23	40	58
	Redclover	Caps	0.47	0.6	0.2	0.4 18.9 16.6 0.00 0.00	37	1	37	40	93
		Tab	0.80	1.6	4.7	5.2 30.2 7.9 0.02 0.00	50	1	50	40	125
		Caps	0.56	1.9	2.9	2.6 24.6 5.5 0.01 0.00	37	1	37	44	84
	Novogen -Redclover exp 2008	Tab	0.48	1.0	0.5	0.6 15.8 23.6 0.00 0.00	41	1	41	40	103
	Novogen-RedClover exp 2005	Tab	0.54	0.8	0.5	0.5 15.2 23.7 0.00 0.00	41	1	41	40	103
	Novogen -Promensil	Tab	0.53	1.0	0.5	0.6 17.1 24.9 0.00 0.00	44	1	44	40	110
		Caps	0.71	1.4	2.6	2.4 36.5 10.5 0.01 0.00	53	1	53	40	133
		Tab	0.54	1.1	0.4	0.4 16.9 25.6 0.00 0.00	44	1	44	40	110
		Tab	0.77	0.3	1.4	1.9 17.7 4.8 0.00 0.00	26	2	52	40	130
		Caps	0.47	0.6	0.1	0.2 24.0 18.6 0.00 0.00	43	1	43	40	108
	Soya and Redclover	Caps	0.35	3.3 23.1 7.7	1.2 0.3 0.00 0.00	36	1	36	62.5	58
		Caps	0.72	7.5 11.7 0.6	0.7 0.5 0.00 0.00	21	1	21	20	105
		Caps	0.72	8.1 12.7 0.6	0.7 0.5 0.00 0.00	22	1	22	20	110
		Caps	0.40	0.9	2.4	0.6	1.2 0.3 0.00 0.00	6	2-3	11	20	55
	Kudzu	Tab	0.55	2.7	6.4	5.8	0.9 0.5 0.04 0.24	16	1	16	35	46
		Tab	1.05	3.9	8.8	8.9	0.2 0.1 0.05 0.14	22	1	22	55	40
		Tab	1.62	1.7	2.8	2.5 31.1 8.3 0.01 0.02	46	1	46	40	115
	Hops	Caps	0.31	1.3	6.1	2.0	0.0 0.0 0.00 0.00	9	1-2	19	17.2	110

*

Gen = genistein, Daid = daidzein, Gly = glycitein, Form = formononetin, Bio = biochanin A, Cou = coumestrol, Pur = puerarin. # Where a dose range is given e.g. 2-3 per day the lower value has been used.

†

The dose is assumed to be measured as the aglycones.

Table 4 .

 4 Differences in quantitation of biochanin A and formononetin in Promensil

	Analyte	Internal std		µg/ml
			Diluted Concentrrated
	Biochanin A	13 C 3 -Genistein	10.6	54
		13 C 3 -Daidzein	7.2	32
	13 C 3 -Biochanin A	12.8	131	Correct value
	13	C 3 -Formononetin	10.3	90
	Formononetin	13 C 3 -Genistein	8.9	48
		13 C 3 -Daidzein	6.1	31
	13 C 3 -Biochanin A	8.7	78
	13 C 3 -Formononetin	10.8	103	Correct value

Table 5 .

 5 Calculation of aglycone equivalents in soya based products

			Molecular	Aglycone	Composition	Aglycone
	Compound		weight	equivalents	ratio	equivalents
	Daidzein	Aglycone	254	1.00	0.01	0.01
		Daidzin	416	0.61	0.52	0.32
		6''-O-Acetyldaidzin	458	0.55	0.09	0.05
		6''-O-Malonyldaidzin	502	0.51	0.38	0.19
					1.00	0.57
	Genistein	Aglycone	270	1.00	0.01	0.01
		Genistin	432	0.63	0.52	0.33
		6''-O-Acetylgenistin	479	0.56	0.09	0.05
		6''-O-Malonylgenistin	518	0.52	0.38	0.20
					1.00	0.58
	Glycitein	Aglycone	284	1.00	0.01	0.01
		Glycitin	446	0.64	0.52	0.33
		6''-O-Acetylglycitin	488	0.58	0.09	0.05
		6''-O-Malonylglycitin	532	0.53	0.38	0.20
					1.00	0.60
			Daidzein/Genistein/Glycitein ratio of 4:1:3	0.58
			Daidzein/Genistein/Glycitein ratio of 7:10:1	0.58

  Addition of 13 C 3 -Daidzein glucuronide before hydrolysis After hydrolysis to 13 C 3 -Daidzein B) Natural daidzein hydrolysed, unspiked With hydrolysis and addition of 13 C 3 -Daidzein glucuronideFigure 4. LC-MS/MS chromatograms of extracts of red clover tablets as a proof of quantitative hydrolysis of glycoside conjugates within the sample matrix. Analysis of samples containing native analyte by concurrent acid hydrolysis of 13 C 3 labelled glucuronide to the 13 C 3 -aglycone.

	Compound name: 13C3 Formononetin Response Factor: 852227 RRF SD: 340931, % Relative SD: 40.0047 Response type: External Std, Area Curve type: RF Compound name: 13C3 Daidzien Response Factor: 75704.8 RRF SD: 21981.6, % Relative SD: 29.0359 Response type: External Std, Area Curve type: RF 0 1 2 3 4 Re sp on se 0 20000 40000 60000 80000 100000 F 5 6 7 o 8 r 9 10 P 11 e 12 13 e 14 r vial no 15 R v Compound name: 13C3 Biochanin A Response Factor: 2.4067e+006 RRF SD: 1.0036e+006, % Relative SD: 41.7004 Response type: External Std, Area Curve type: RF Compound name: 13C3 Genistein Response Factor: 94677.8 RRF SD: 29404.4, % Relative SD: 31.0574 Response type: External Std, Area Curve type: RF 0 1 2 3 4 5 Res pon se 0 20000 40000 60000 80000 100000 120000 e i 5797.469 6 7 5.80e3 19903 MM 13C3-Daidzein 16.48 e % 2 Daidzein 16.48 13410.122 1.34e4 49382 MM 15000.000 A) MRM of 22 channels ,ES-vial no 8 9 10 11 12 13 14 15 17.77 256 > 226 2.131e+004 m in MRM of 22 channels ,ES-253>223 5.111e+004 17.77
		1000000 1200000																			2500000 3000000 w
	Re	800000																		Re
	sp on se	0 200000 400000 600000	0	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	Vial no	sp on se	0 500000 1000000 1500000 2000000	0	1	2 O 3 4 5 n 6 l 7 8	9	10	11	12	13	14	vial no 15
																								y
																			3

Acknowledgments

work was provided in part by Novogen Limited.