atom of interest ( I , 5, 8). We have attempted a correlation here by calculating the charge on the atom by the Pauling method (9), and plotting this us. observed binding energy (Figure 2A). In order to calculate the charge, we have assumed that the effect of atoms farther down the carbon chain than the adjacent carbon and atoms bound to it is negligible. Inspection of Figure 2A shows that this approach underestimates the effect of substitution on the adjacent carbon. This has been observed previously with ligand effects on binding energies in platinum complexes (8). In that case a simple method was developed for estimating effective group electronegativities so that the sum of substituent electronegativities could be correlated with observed binding energies. We have adapted the method described in Reference 8 to polymers and achieved the correlation shown in Figure 2B. According to this method, substituent group electronegativities are taken to be simply the arithmetic mean of the electronegativities of the constituent atoms. The summing is limited, however, to the central atom of the group and the atoms attached directly to it. Substituent groups which are actually part of the chain are given only ’/* weight in the summation since they are in turn part of the next unit down the chain. Thus for example when the sum of substituent electro(8) W. M. Riggs, ANAL.CHEM.,44,390 (1972). (9) L. Pauling, “The Nature of the Chemical Bond,” 3rd ed., Cornel1 University Press, Ithaca, N.Y., 1960.
negativities was calculated for -CH2- carbon in -(CF2 -CH&, the atoms shown below were considered. F H F
c-c-I cl -c-c l I
l
l
F H F
The carbon in question is considered to have four substituents’: 2 hydrogen atoms and 2 -CFz-C groups. However, the calculated -CF2-C group electronegativity is given only half the weight of a complete group since it in turn is part of the next unit down the polymer chain. An equivalent way of regarding this is to consider that the basic unit is [-(CCFz-) -CH2-] and there are thus only three substituent electronegativities to include in the sum. It is not immediately apparent on theoretical grounds that this approach should yield such good correlations. However, Thomas (5) has offered plausible rationalizations for this, at least for simple systems. This approach yields results equivalent to or better than the Pauling method through simpler arithmetic manipulations, and having succeeded for two quite different kinds of systems, may have some degree of general applicability for making quick estimates of direction and magnitude of binding energy shifts in chemical systems of interest. RECEIVED for review January 3, 1972. Accepted March 1, 1972.
Quantitative Liquid Chromatography of Sulfonylureas in Pharmaceutical Products William F. Beyer Control Analytical Research and Development; The Upjohn Company, Kalamazoo, Mich. 49001
THE SULFONYLUREA ANTIDIABETIC AGENTS (see Table I for structures) are usually determined by ultraviolet spectrometry or potentiometric titration ( I , 2). To increase specificity, thin layer chromatography has also been applied to the assay of these compounds (3-8). Attempts to develop gas liquid chromatographic (GLC) procedures for intact or derivatized intact sulfonylureas have been largely unsuccessful, primarily because of the thermal liability of these compounds at the temperatures required for chromatography. Utilizing thermal fragmentation to p toluenesulfonamide, a GLC assay for tolazamide has been
developed recently by Wickramasinghe and Shaw (9). Sabih and Sabih reported a GLC method for the determination of tolbutamide and chlorpropamide after derivitization with dimethyl sulfate (10). However, in our laboratories, we have not been able to chromatograph tolbutamide as a single peak using this procedure. Although the N-methyl derivative of tolbutamide was the principal product, approximately 10% of the methylenol ether was also formed (11). High-speed liquid chromatography has provided a highly specific and practical technique for the analysis of these antidiabetic agents, and is the subject of this report. EXPERIMENTAL
“United States Pharmacopeia.” 18th. rev., Mack Publishing Co., Easton, Pa., 1970. (2) “The National Formulary,” 13th ed., Mack Publishing Co., Easton, Pa. 1970. (3) K. C . Guven, S. Geegil, and 0. Pekin, Eczacilik Bul., 8, 158 (1966). (4) M. G. Hutzul and C. F. Wright, Can. J. Pharm. Sci., 3, 4 (1968). ( 5 ) D. L. Smith, T. J. Vecchio, and A. A. Forist, Metabolism, 15 (3), Part I (March). 1965. (6) J. Baumler and S. Rippstein, Deut. Apoth.-Zg., 107, 1647 (1967). (7) 0. Schettino and M. I. LaRotonda, Boll. SOC.Ital. Giol. S per. 1970, 46 (8), 432-6. (8) Ibid., pp 436-8. (1)
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ANALYTICAL C H E M I S T R Y , VOL. 44, N O . 7, J U N E 1972
Apparatus. A DuPont model 820 liquid chromatograph operated at ambient temperature with a UV detector at 254 nm was used. The column was stainless steel, 100 cm long and 2.1 mm internal diameter. It was dry-packed with a hydro-carbon polymer support (1 ethylene propylene copolymer on DuPont Zipax, catalog. No. HCP-820960008, The DuPont Company, Wilmington, Del.). Peak areas were ~
(9) J. A. F. Wickramasinghe and S. R. Shaw, J . Pharm. Sci., 60, 1669 (1971). (10) K. Sabih and K. Sabih, ibid., 59 (6) 1970. (11) P. Bowman, The Upjohn Co., Kalamazoo, Mich., private communication 1971.
1.50
-
1.25
-
/ v)
c z
1.00 1.00
a
I
Figure 1. Chromatogram for glyburide and internal standard (testosterone) and plot for glyburide us. peak area ratios using the internal standard at 0.25 mg/ml
Y
u
z
3 a 0
0.50
v)
m
a
0.2
0.00 0 0
I
I I 0.8 I I 1.2 I I 1.6 I 0.4 0.4 0.8 1.2 1.6 GLYBURIDE (mcg)
I
0
4
8
12 MINUTES
16
. ~
Table I. Nomenclature and Structural Formulas for Sulfonylurea Antidiabetic Agents GENERIC NAME
S T R U C T U R A L FORMULA
Cl
GLYBURIDE
CHLORPROPAMIDE
C
TOLAZAMIDE
c
l
H
e S02NHCONMCb12CH3
3
Table 11. Chromatographic Data for Sulfonylureas Flow Retention Elution hdobile rate. time, volume, Compound phase ml/min minutes ml Glyburide Ia 0.25 8.5 2.1 IIb 0.36 6.5 2.3 Chlorpropamide I1 0.36 7.5 2.7 Tolazamide I1 0.36 9.0 3.2 Tolbutamide 11 0.36 11.3 4.1 Acetohexamide 0.01M sodium borate containing 27.5% methanol. 0.01M monobasic sodium citrate containing 15 % methanol. Q
0S q N H C O N K N g
tablets were ground to a fine powder. Accurately weighed amounts of powdered tablets, equivalent to the approximate quantities of the sulfonylureas indicated, were extracted with 20.0 ml of the appropriate Internal Standard Preparation: glyburide, 5 mg with A ; tolbutamide, 70 mg with B; tolazamide, 80 mg with C; acetohexamide, 40 mg with B; and chlorpropamide, 40 mg with C. The extractions were carried out in 7-dram disposablevials, securely closed with polyethylene caps. After vigorous shaking for approximately 30 minutes, the samples were centrifuged for about 3 minutes. A further dilution of the acetohexamide sample preparation was made by diluting 2.0 ml of the clear solution to 25.0 ml with B. CHROMATOGRAPHY. Glyburide was chromatographed using mobile Phase I at a flow rate of 0.25 ml. Tolbutamide, tolazamide, acetohexamide, and chlorpropamide wzre chromatographed using Mobile Phase I1 at a flow rate of 0.36 ml/ min. The column and mobile phases were operated at 500 psig and at room temperature. Approximately 1 p1 of standard and sample preparations were injected on the column. The amount of drug in each tablet was CALCULATIONS. calculated by the internal standard-reference standard method.
integrated with a n electronic integrator (Model 6210, Vidar, Mountain View, Calif.). Reagent and Solutions. MOBILEPHASES.Mobile Phase I was 0.01M sodium borate containing 27.5 methanol (v/v), a t a n apparent p H of 9.2. Mobile Phase I1 was 0.01M monobasic sodium citrate containing 15 methanol (v/v) a t a n apparent p H of 4.4. The citrate buffer was prepared from reagent grade citric acid and sodium hydroxide and made fresh daily. INTERNAL STANDARDSOLUTIONS.The following compounds (chromatographically pure) were dissolved in 3A alcohol (95 % ethanol, denatured with approximately 5 methanol, U S . Industrial Chemicals Company, New York, RESULTS AND DISCUSSION N.Y.) to give solutions of the following concentrations: A. testosterone, 0.125 mg/ml; B. chlorpropamide, 1.80 mg/ml; When Mobile Phase I was used at a flow rate of 0.25 ml/ and C. acetohexamide, 0.150 mglml. minute, glyburide was eluted at an elution volume of 2.1 ml STANDARDPREPARATIONS. The following compounds (retention time of 8.5 min). Figure 1 gives a typical chrowere dissolved in the internal standard solutions (A, B, or C) matogram for glyburide and the internal standard, tesat the concentrations indicated: Upjohn Glyburide Refertosterone. The small peak just prior to the glyburide peak was ence Standard, 0.50 mg/ml in A ; USP Tolbutamide Reference identified as 5-chloro-N-(p-sulfamoyl phenethy1)-o-anasimide, Standard, 3.50 mg/ml in B: USP Tolazamide Reference Stana degradation product of the sulfonylurea. The figure also dard, 4.00 mg/mI in C ; N F Acetohexamide Reference gives a linearity plot for various concentrations of glyburide Standard, 0.160 mg/ml in B; and USP Chlorpropamide Refercs. peak area ratios of the compound and internal standard. ence Standard, 2.00 mg/ml in C. Procedure. COMPRESSED TABLETSAMPLEPREPARATION. Chlorpropamide, tolazamide, tolbutamide, and acetohexThe average weight of 10 tablets was determined and the amide were eluted at a flow rate of 0.36 ml,'min using Mobile
x
ANALYTICAL CHEMISTRY, VOL. 44, NO. 7, J U N E 1972
0
1313
I S
t
On016
t
0.008
v)
L
a I
IV
u Ly
L
nGlyburide
Solvent
h I'l ii
om 9-Tolbutamide
0.004 5
10
5 15 MINUTES
10
15
Figure 2. Liquid chromatograms of sulfonylureas at a flow rate of 0.36 ml/min A . mobile phase = 0.01 monobasic sodium citrate containing 15 methanol; B. mobile phase = 0.01M monobasic sodium citrate containing 10 methanol Key: S, solvent; I, chlorpropamide, 1.50 p g ; 11, tolazamide, 2.50 pg; 111, tolbutamide, 2.50 p g ; and IV, acetohexamide, 0.25 pg
Phase 11. Table I1 gives elution volumes and retention times for the compounds. Linearity of peak area ratios 6s. concentrations of the sulfonylureas was tested by chromatographing five to six concentration levels of each sulfonylurea dissolved in the appropriate internal standard solutions (see Figure 2). The correlation coefficients did not differ significantly from 1.0. Figure 3 shows the effect of varying the methanol concentrations in the mobile phase. Base-line resolution for a mixture of chlorpropamide, tolazamide, tolbutamide, and acetohexamide was obtained with 10% methanol in the mobile phase. A higher concentration of methanol (15%) gave adequate resolution for quantitation of the individual sulfonylureas and shortened chromatographic time. Two of the sulfonylureas, chlorpropamide and acetohexamide, were used as internal standards for purposes of quantitation. These compounds were used as internal standards because of their excellent resolution from the sulfonylureas being tested, and because internal standards are often preferred that are structurally similar to compounds tested. Based on peak area integrations relative to amounts of compounds chromatographed, response ratios at 254 nm for chlorpropamide : tolazamide :tolbutamide:acetohexamide were 2.1 : 1.0:1.1 : 23.0, respectively. Chromatography of the sulfonylureas was aided by modifying the methanol with acidic and alkaline buffers. The elution volumes for all of the sulfonylureas investigated decreased as the pH of the mobile phase was raised. This behavior parallels the solubility in the mobile phase of these compounds, which being weak acids, are more soluble in solutions of higher pH. Various compounds structurally related to the sulfonylureas, and which could be present under normal circumstances as impurities or degradation products, did not interfere in the assays under the conditions used.
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ANALYTICAL CHEMISTRY, VOL. 44, NO. 7, J U N E 1972
0.000
0
5
10 15 MINUTES
20
25
Figure 3. Chromatograms of glyburide and a degradation product (Mobile phase = 0.01 Msodium borate containing 27.5% methanol; flow rate = 0.1 ml/min) and tolbutamide and its o-methyl isomer (Mobile phase = 0.01M monobasic sodium citrate containing 7.5 % methanol; flow rate = 0.36 ml/min Figure 3 demonstrates that base-line resolution was obtained for a possible impurity in glyburide using a flow rate of 0.10 ml/min for Mobile Phase I. This compound, 5-chloro-N(p-sulfomoylphenethy1)-o-anasimide,was quantitated at a level of 2% or less, using either pure reference material or a response factor. The figure also demonstrates base-line resolution for tolbutamide and its ortho-methyl isomer with 7.5 % methanol in the mobile phase. The recovery of the sulfonylureas from inert tablet ingredients was tested and was near 100%. For the glyburide study, the equivalent of one placebo tablet as a powder mixture was added to an appropriate volume of testosterone internal standard preparation containing various amounts of glyburide standard. To determine recovery for chlorpropamide, tolazamide, tolbutamide, and acetohexamide, weighed amounts of drug standards and the appropriate powdered tablets were mixed and added to the internal standard solutions. The concentrations of the drugs were determined by assay against the appropriate standard, using peak area ratio calculations. Recoveries of the added drug standards were quantitative, varying from 98.9 % for acetohexamide to 100.2% for tolazamide. Precision was determined by assaying at least six samples of powdered tablets from single lots of glyburide, chlorpropamide, tolazamide, tolbutamide, and acetohexamide. The relative standard deviation ranged from 0.8% for tolbutamide tablets to 2.56% for tolazamide tablets. Tablet assays were carried out for four lots from each of the commercially available sulfonylureas of this report. All lots were within requirements of NF and USP for these tablets. RECEIVED for review October 26, 1971. Accepted January 7 , 1972.
