table 1______________________________________amounts required for a 5 l batchingredient amount amount per ml______________________________________ceftiofur hcl micronized 0 . 258 kg * 50 mgphospholipon - 90h 0 . 25 g 0 . 5 mgsorbitan monooleate nf 0 . 75 g 1 . 5 mgwater for injection , usp 1 . 25 g 2 . 5 mgcottonseed oil q . s . to 5 l q . s . to 1 ml______________________________________ * the amount is of active ceftiofur taking into consideration the salt . assumes 100 % potent ceftiofur hcl with 3 . 2 % w / w of the weight accounting for the hcl . mw of ceftiofur hcl is 560 . 0 . the required weight or volume of oil is placed into a glass or stainless steel vessel . ( see table 1 for the amounts required for a 5 l batch .) for a 5 l batch , 4 . 5 l of cottonseed oil is used to begin . the oil is heated to above 100 ° c . and the required amount of phospholipon ( lecithin ) is added and stirred until dissolved . the time required for the phospholipon ( lecithin ) to dissolve depends on the temperature , mixing , and size of the batch . it normally dissolves within 1 to 60 minutes . the oil containing the phospholipon is then cooled . once cooled the sorbitan monooleate is added and mixed . next the ceftiofur hcl is added followed by mixing of 1 to 120 minutes . the length of mixing depends on the batch size , size of mixer and speed of mixing . water is added and the suspension is mixed for 1 to 60 minutes . the suspension is stored in the original manufacturing vessel as long as mixing continues to keep the drug suspended . it is then filled into vials using standard vial filling equipment . the vials are then closed with a stopper , capped , labeled and boxed . comparison of the currently marketed formulation of ceftiofur hydrochloride and formulations of the present invention one of the most important differences observed between the fomulations was the greater physical stability or resuspendability when water was added to the formulation . shown in fig1 is a plot of resuspendability compared to the water content of various ceftiofur hcl suspensions . it is clear from this figure that as the water content increased , the suspension resuspended better . the resuspendability of suspensions with lower water content was poor or , at best , variable . it was not until the water content was increased that consistently more resuspendable suspensions were achieved . more specific physical stability comparisons are shown in the next section . several other differences were observed when increasing the water content of the suspension . all these differences help explain why greater physical stability was observed when adding water : first , sedimentation volumes were greater , and the sedimentation rates faster when water was added . the sedimentation volume is the height of the sediment when compared to the height when the suspension is fully resuspended . larger sedimentation volumes typically are associated with a suspension that resuspends better . less packaging of the sediment occurs making it easier to resuspend ( i . e ., less energy needs to be put into the system via shaking ). second , the faster settling rate indicates the particles are interacting to create a flocculated system . a flocculated suspension typically resuspends better then a nonflocculated suspension . the flocs that form will be larger then the original particles so they settle faster . but , because they interact with themselves and other flocs , they will not settle to such low sedimentation volumes . thus , they are easier to resuspend . the addition of water to the formulation of the present invention caused flocculation and faster sedimentation rates were observed . also , flocculation was observed with particle size data . larger measured particles were observed as the water content of the suspension increased . furthermore , photomicrographs were taken showing the particles interacting to create larger flocs . finally , rheology differences were noted when adding water to the formulation . the rheology , or flow characteristics , changed from newtonian to non - newtonian with added water . the plastic systems found with added water indicate a structure was formed in the water - added suspensions . this added structure again indicates a flocculated system which will have improved resuspendability . in summary , all of the differences found between the suspensions with and without added water indicate that physical stability will be better with the water - added formulation . the stability of suspensions , and ultimately their shelf - life , are based on both chemical and physical stability . the chemical stability is assessed to insure that the product does not become subpotent during use . the chemical stability of the current formulation ( ie . non - water - added formulation ) has been studied extensively . for example , shown in table 2 is the stability through 3 years for 3 different batches of the current suspension . table 2______________________________________chemical stability of a 50 mg / ml ceftiofur hcl suspension with noadditional water added when stored at 25 ° c . potency in mg / ml ± standard deviationtime , months lot a lot b lot c______________________________________ 0 49 . 8 ± 0 . 7 51 . 1 ± 0 . 5 50 . 5 ± 0 . 3 3 48 . 4 ± 1 . 3 49 . 4 ± 1 . 6 48 . 4 ± 0 . 4 6 48 . 1 ± 0 . 7 48 . 8 ± 1 . 2 48 . 6 ± 0 . 712 49 . 1 ± 0 . 4 50 . 2 ± 0 . 3 50 . 1 ± 0 . 318 46 . 5 ± 1 . 4 48 . 5 ± 0 . 7 48 . 5 ± 0 . 924 46 . 8 ± 4 . 2 48 . 8 ± 0 . 6 49 . 2 ± 2 . 736 49 . 1 ± 0 . 8 49 . 5 ± 0 . 4 48 . 6 ± 0 . 7______________________________________ usually pharmaceutical products are allowed to decrease 10 % in potency during their shelf - life . table 2 shows the current suspension is chemically stable even out to three years when stored at room temperatures . the chemical stability has not been assessed for as long with the formulation of the present invention containing additional water . shown in table 3 is the comparison of two batches that were made identically except for the amount of water added . similarly , table 4 shows another comparison of two batches that only varied in water content . table 3______________________________________comparison of chemical stability of two batches with different watercontents when stored at 25 ° c . potency as a percent of label ± standard deviationtime , months lot d ( 0 . 20 % total water ) lot e ( 0 . 39 % total water ) ______________________________________0 103 . 2 ± 0 . 5 102 . 2 ± 0 . 22 103 . 2 ± 0 . 4 101 . 6 ± 0 . 04 103 . 6 ± 0 . 3 103 . 3 ± 1 . 36 103 . 7 ± 0 . 4 103 . 0 ± 0 . 2______________________________________ table 4______________________________________comparison of chemical stability of two batches with different watercontents when stored at 25 ° c . potency as a percent of label ± standard deviationtime , months lot f ( 0 . 14 % total water ) lot g ( 0 . 67 % total water ) ______________________________________0 104 . 5 ± 0 . 3 103 . 4 ± 0 . 62 104 . 6 ± 1 . 7 103 . 1 ± 0 . 14 105 . 5 ± 0 . 7 105 . 4 ± 0 . 56 103 . 5 ± 0 . 5 102 . 9 ± 0 . 1______________________________________ both tables 3 and 4 show the suspension to be chemically stable . no difference was observed in chemical stability even with high water content . thus , contrary to what may be expected , the addition of water did not adversely affect the chemical stability of the suspension . additional stability data which was obtained at accelerated temperatures shows no difference in stability with higher water content . physical stability is just as important as chemical stability . if the product does not resuspend adequately when shaken , the dose will be incorrect . with incomplete resuspension when shaken , the initial doses removed will be subpotent . this occurs because drug remains at the bottom of the vial preventing the correct concentration of the suspension when agitated . if part of the product is removed at a concentration significantly less then the labeled concentration , doses removed when the vial contains less product could become superpotent . this happens when further agitation removes drug from the bottom of the container and it is dispersed in the smaller volume of liquid . the current formulation ( ie . no water added ) needs to be shaken , sometimes up to 60 seconds , before it becomes fully resuspended as shown in table 5 : table 5______________________________________physical stability of a 50 mg / ml ceftiofur hcl suspension with noadditional water added when stored at 25 ° c . time required to fully resuspend , secondstime , months lot a lot b lot c______________________________________ 0 10 - 20 10 10 3 30 - 40 10 - 20 20 - 30 6 30 10 2012 50 - 60 20 - 30 40 - 5018 40 - 60 20 3024 50 - 60 60 40 - 6036 60 40 - 50 60______________________________________ it has been found that the addition of water improves the resuspendability characteristics of the suspension . a sensitive method for monitoring resuspendability is to use a 10 - second mechanical shake assay . in this test , a vial is shaken for 10 seconds using a mechanical arm . a sample is removed from the suspension 2 cm below the liquid / air interface and assayed for the amount of drug . shown in tables 6 and 7 below are batches produced by identical methods except for the amount of water : table 6______________________________________comparison of physical stability of two batches with different watercontents using a 10 - second mechanical shake test potency as a percent of label ± standard deviationtime , months lot d ( 0 . 20 % total water ) lot e ( 0 . 39 % total water ) ______________________________________0 82 ± 4 99 ± 22 72 ± 40 98 ± 14 48 ± 7 97 ± 26 49 ± 3 96 ± 1______________________________________ table 7______________________________________comparison of physical stability of two batches with different watercontents using a 10 - second mechanical shake test potency as a percent of label ± standard deviationtime , months lot f ( 0 . 14 % total water ) lot g ( 0 . 67 % total water ) ______________________________________0 79 ± 5 104 ± 12 32 ± 21 101 ± 24 58 ± 16 102 ± 36 67 ± 27 95 ± 2______________________________________ as the data from these tables shows , the increased water content improved resuspendability . the formulations with higher water concentrations were closer to the theoretical 100 % of label and had less vial - to - vial variability ( ie . tighter standard deviations ). fig1 is a plot of resuspendability compared to the water content of various ceftiofur hcl suspensions .