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Betamethasone Sodium Phosphate

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Betamethasone Sodium Phosphate ci = free (unbound) molar concentration of ith compound SO = s evaluated at t = 0 c = (c1, cp, . , CM)~(see cj) t = time [c,] = total (free + bound) molar concentration of ith compound T = transpose [cl = ([ell, [czl,. , [cMI)~(see [cJ ui = molecular weight of ith compound (Cb)r = “membrane bound concentration” of ith compound u = subscript (u = 1,2,. .) D = diag(l/KI, 1/Kz, . , I/KM) = diagonal matrix containing V = volume of dialysis compartment reciprocals of dialysis rate constants w = weight of available membrane material f, = function describing relationship between [ci]and c or [ci]and yi = ith dependent variable in transformed set of linear regression S equations (Eq. A2) used to obtain initial estimates of the f = (/I, fz, . , ~MM)~(see fi) binding parameters (n’sand k’s) by multiple linear regression gi = function describing relationship between (cb), and s, technique g = (gi, gz, . , gu)T(seegi) REFERENCES i = subscript (i = 1,2,. .) j = subscript (j= 1,2,. .) (1) H. H. Stein, Anal. Biochem., 13,605 (1965). A. J - = Jacobian matrix off with respect to s (2) Agren, Acta Pharm. Suec., 5.37 (1968). <I> (3) M. C. Meyer and D. E. Guttman, J. Pharm. Sci., 57, 1627 J,, = ijth element of Jacobian matrix (1968). ki, = association constant for ith compound’s binding to jth class (4) Ibid., 59,33 (1970). of binding sites on macromolecule (5) Ibid., 59,39 (1970). ki = kil (see ki;) (6) J. C. Dearden and E. Tomlinson, J. Pharm. Pharmacol., Suppl., k;* = membrane binding “association constant” of ith compound 22,53S (1970). K, = dialysis rate constant for mth compound (7) M. J. Crooks and K. F. Brown, J. Pharm. Sci., 62,1904 (1973). m = subscript (m = 1,2,. .) (8) M. J. Crooks and K. F. Brown, J. Pharm. Pharmacol., 26,235 A4 = number of compounds (1974). n, = number of binding sites in jth class of sites (9) I. Kanfer and D. R. Cooper, ibid., 28,58 (1976). n;* = mathematical analog to ni in Langmuir-type membrane (10) S. J. A. Kazmi and A. G. Mitchell, J. Pharm. Sci., 62, 1299 “binding” model (1973). n = nl (seen,) (11) F. Bottari, G. Di Colo, E. Nannipieri, M. F. Saettone, and M. F. N = number of binding classes Serafini, ibid., 64,946 (1975). V, = number of moles of ith compound (“ligand”) bound per mole (12) P. Veng Pedersen, M. J. Crooks, and K. F. Brown, J. Pharm. Sci., of macromolecule 66,1458 (1977). &* = amount of ith compound bound per amount of available (13) C. W. Gear, “Numerical Initial Value Problems in Ordinary membrane material Differential Equations,” Prentice-Hall, Englewood Cliffs, N.J., 1971. P = total molar concentration of macromolecular compound (14) “Handbook of Mathematical Functions,” M. Abramowitz and 0 = parameter vector containing elements such as nj, ki;, and P I. A. Stegun, Eds., Dover, New York, N.Y., 1970, p. 897. or other parameters used in the particular mathematical (15) Ibid., p. 950. model f describing binding kinetics (16) P. Veng Pedersen, J. Pharmacokinet. Biopharm., 5, 513 r = correlation coefficient (1977).\--. .,- s, = absolute value of slope in a [ci] uersus t plot at time t. This (17) Y. Bard, “Nonlinear Parameter Estimation,” Academic, New quantity is used as a parameter variable in the parametric York, N.Y., 1974, p. 57. representation of the variables [ci] and t (18) J. H. Ahlberg, E. N. Nilson, and J. L. Walsh, “The Theory of s = (S~,SP,.. , SM)T(see si) Splines and Their Applications,” Academic, New York, N.Y., 1967. Analysis of Steroid Phosphates by High-pressure Liquid Chromatography: Betamethasone Sodium Phosphate L. M. UPTON *, E. R. TOWNLEY x, and F. D. SANCILIO * Received July 18,1977, from the Physical and Analytical Chemical Research and Development Department, Schering Corporation, Bloomfield, NJ 07003. Accepted for publication October 25,1977. *Present address: Seton Hall University, South Orange, NJ 07079. $Present address: Burroughs Wellcome Co., Research Triangle Park, NC 27709. Abstract A sensitive, automatable high-pressure liquid chromato- chromatographic analysis in dosage forms High-pressure liquid graphic procedure is presented for the determination of steroid phos- chromatography-analysis, betamethasone sodium phosphate in dosage phates. Quantitation is described for betamethasone sodium phosphate forms Glucocorticoids-betamethasone sodium phosphate, high- in dosage forms in the presence of polar excipients. The separation of a pressure liquid chromatographic analysis in dosage forms Steroid multicomponent mixture of steroid phosphates also is reported. phosphates, various-high-pressure liquid chromatographic analysis in dosage forms Keyphrases 0 Betamethasone sodium phosphate-high-pressure liquid Steroid phosphates are highly effective anti-inflam- reaction methods (1-3). These procedures are time con- matory agents produced in ophthalmic, injectable, and suming and relatively difficult. Two chromatographic solid dosage forms. Reported analyses utilize spectro- methods have been reported: TLC of the methyl ester and photometric procedures preceded by extraction and/or ion chromatography (4,5). This laboratory previously used 0022-3549/ 78l0700/0913$01.00/0 Journal of Pharmaceutical Sciences / 913 @ 1978, American Pharmaceutical Association Vol. 67, No. 7, July 1978 Table I-Retention Times for Betamethasone Sodium Table 11-Method Reproducibility a Phosphate, Its Decomposition Products, and Common Preservatives Used in Formulations Betamethasone Sodium Phosphateb, mg/ml Retention Sample Assay I Assay Compound Time", min I1 3.8 3.9 Decomposition product 2.5-3.5 3.8 3.9~. Phenol 4.0 3.7 3.7 Betamethasone sodium --phosphate 5.0 3.7 3.8 Decomposition product 5.5 3.8 3.8 Betamethasone alcohol 8.0 Decomposition product 11.0 Butylparaben 12.0 Mobile phase: methanol-0.09 M KHzPOd (60:40). Each assay result is an Methandrostenolone 15.0 average of two injections. 0 Mobile phase: methanol-0.09 M KH2P04 (1:l). The peak height ratios were used to quantitate the resulting chro- paper chromatography followed by elution of the steroid matograms. Equation 1 was used to calculate the response factor (RF) for the reference standard versus the internal standard: phosphate and quantitative measurement by colorimetric assay or by an enzymatic-colorimetric analysis. RF= (z) (0.133) (Eq. 1) The present study was undertaken to establish a rapid, (g) reliable, stability-indicating high-pressure liquid chro- where PHI is the peak height or area of betamethasone sodium phosphate in the response factor standard solution, PH2 is the peak height or area matographic (HPLC) procedure for steroid phosphates, of butylparaben in the response factor standard solution, W;, is the weight specifically for betamethasone sodium phosphate in the (in milligrams) of butylparaben in the internal standard solution, W, is presence of compounds typically present in formulations. the corrected weight (in milligrams) of standard in the reference standard Quantitation of betamethasone sodium phosphate and solution, and 0.133 is the combined dilution factor of sample and internal other steroid phosphates in various formulations was standard solutions (l/60 X l0/1 X z/8 X 8/3). This calculation is repeated for each determination, and an average achieved. response factor is used to derive the milligrams of betamethasone sodium phosphate per milliliter of original sample using: EXPERIMENTAL mg/ml= (-)PH3 (g)(i) (0.04) Apparatus-A liquid chromatograph' with a UV absorption detector PH4 (254 nm) and a stainless steel column, 30 cm X 4 mm i.d., was used. The where PH3 is the peak height or area of betamethasone sodium phosphate column packing2 was porous silica particles permanently bonded to a in the sample solution, PH4 is the peak height or area of butylparaben monomolecular layer of organosilane. The column was operated at 1200 in the sample solution, RF is the average response factor, V is the volume psi, resulting in a flow rate of 1 ml/min. Each analysis was performed at of sample used in the preparation of the sample solution, and 0.04 is the ambient temperature. Samples were injected with a 5-pl high-pressure combined dilution factor of sample and internal standard solutions ('/so syringe3. x 218 x 8/d. Materials-Acetone', methanol4, b~tylparaben~,and methandro- Determination of Corrected Standard Weight, WS8-Reference stenolone5 were used as received. The samples of betamethasone sodium standard solution (q.0 ml) is diluted with water to 200 ml in a volumetric phosphate, prednisolone sodium phosphate, dichlorisone sodium phos- flask. The absorbance (A,)is read at 240 nm; W,, in milligrams, is cal- phate, testosterone sodium phosphate, and 16P-methylprednisone so- culated in accordance with: dium phosphate were obtained in pure form5. Mobile Phase-The mobile phase was degassed methanol-0.09 M W, = (A,) (33.67) (Eq. 3) KHzPO~~(6040) (Fig. 1). Multicomponent steroid phosphates were separated using methanol-0.09 M KH2P04 (1:l) (Fig. 2). Internal Standard, Reference Standard, and Sample Solu- tions-The internal standard solution of butylparaben was prepared by dissolving an accurately weighed (-150 mg) quantity of material in ac- etone in a 50-ml volumetric flask. The reference standard, about 35 mg of betamethasone sodium phosphate, was weighed accurately into each of two 10-mlvolumetric flasks and dissolved in water. A response factor solution was prepared by adding 3.0 ml of reference standard solution, 2.0 mi of internal standard solution, and 3.0 ml of water to a stoppered flask. The remaining quantity of reference standard solution was saved for the corrected reference standard weight determination described under Determination o/ Corrected Standard Weight. The sample solution consisted of an aliquot of sample equivalent to about 10 mg of betamethasone sodium phosphate", 2.0 ml of internal standard solution, and water (total of 8.0 m1)8. Chromatography-Each of two analyses consisted of an injection of 5 pI of sample solution followed by a 5-pl injection of a reference standard solution into an equilibrated column, using a detector attenuation of 0.64 absorbance unit.
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