Anal. Chem. 1980, 52, 1368-1370
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change the amplitude of the pulses and its cost is not included. T h e layout of high-speed circuitry is very important for optimum performance. The layout and further information about this circuit may be obtained from the authors upon request. ACKNOWLEDGMENT The authors are grateful to William Saxton, NOAA Aeronomy Laboratory, Boulder, Colo., for his advice on the design of the pulse pair generator.
LITERATURE CITED (1) Borders, R. A,; Birks, J. W.; Borders, J. A. Anal. Chem., article in this issue.
RECEIVED for review October 1,1979. Accepted April 11,1980. This work was performed as partial fulfillment of the requirements for the Ph.D. degree (R.A.B.) in the Department of Chemistry, School of Chemical Sciences, University of Illinois a t Urbana-Champaign, Urbana, Illinois 61801.
High Performance Liquid Chromatographic Determination of Panthenol in Bulk, Premix, and Multivitamin Preparations Herminia Umagat and Ronald Tscherne Research and Diagnostic Products Section, Quality Control Department, Hoffmann-La Roche Inc., Nutley, New Jersey 071 10
Panthenol, 2,4-dihydroxy-N-(3-hydroxypropyl)-3,3-dimethylbutyramide, exists in two optically active forms, both of which are used in pharmaceutical preparations. Chemical, physicochemical, polarographic, and microbiological methods have been used for the determination of panthenol (1-3). In all the methods reported, preliminary sample cleanup was necessary to eliminate interfering substances, a procedure which is often tedious and can result in error due to incomplete recovery. Either ion-exchange chromatography or selective precipitation ( 4 ) has been employed to separate panthenol from interfering compounds which are present in multivitamin preparations, such as vitamins B2 and Be, followed by polarographic determination of the panthenol. Fluorometric determination of panthenol in multivitamins using ninhydrin ( 5 ) requires sample pretreatment which is accomplished by crystallization of the sugar in the matrix with ethanol, extraction from the dry residue with chloroform, and purification of the extract on a chromatographic column packed with ion-exchange resin. Several assays with internal standards have to be performed t o standardize the experimental conditions for the recovery of panthenol from the chromatographic column. Colorimetric determinations using ninhydrin and 1,2naphthoquinone-4-sulfonate (6),hydroxylamine-sodium hydroxide (7), concentrated sulfuric acid (8),and iodine (9) are based on the fact that panthenol undergoes acid or alkaline hydrolysis and forms products that can be determined using a suitable color reaction. The only exception to this is the colorimetric determination of the hydroxamic acid derivative which employs complex formation with iron(II1) ions (10). A nonaqueous titration with perchloric acid has been used for the determination of panthenol but is applicable only to bulk material (11). This paper describes a high performance liquid chromatographic (HPLC) method for the determination of panthenol in bulk and pharmaceutical preparations. In comparison to chemical methods reported, it is faster and more specific and also can be employed to monitor trace levels of aminopropanol, a precursor to panthenol. EXPERIMENTAL Apparatus a n d Conditions. A Waters Associates Model 6000A solvent delivery system was used to pump mobile phase through a Chromegabond C-18 (4.6 mm i.d. X 30 cm) column at a flow rate of 1 mL/min. A Waters Model U6K sample injector 0003-2700/80/0352-1368$01 .OO/O
and an LDC Spectromonitor I1 Model 1202 UV-VIS absorbance detector set at 390 nm were used for the analysis of all samples. Spectra of the panthenol-fluorescamine derivative were recorded using a Cary 14 recording spectrophotometer. Mobile Phase. Mobile phase was prepared by mixing 300 mL of methanol with 700 mL of 0.1 M borate buffer which was adjusted to pH 8.0 f 0.1 with 2 N sodium hydroxide. Reagents, Samples and Standards. Acetonitrile, methanol, 37.7% hydrochloric acid, sodium hydroxide, and boric acid were all ACS reagent grade. c-Aminocaproic acid was purchased from Calbiochem-Behring Corp., La Jolla, Calif. Aminoethanol was purchased from the Aldrich Chemical Co., Metuchen, N.J. Fluorescamine, pathenol reference standard bulk samples, 33% d-panthenol, Vi Penta Multivitamin Drops, and Berocca-C Vials were obtained from Hoffmann-La Roche Inc., Nutley, N.J. Sample and Reference Standard Preparation. An equivalent of from 10 to 20 mg of panthenol was dissolved in 10 mL of 0.5 N HCl and was hydrolyzed at 85 f 2 "C for 30 min in a Fisher Versa-Bath. An aliquot containing 1-2 mg panthenol was transferred into a 25-mL volumetric flask to which was added 10 mL of fluorescamine solution in acetonitrile (concentration of 0.4 mg/mL) and 2-mL of c-aminocaproicacid solution dissolved in the mobile phase (concentration approximately 60% that of panthenol) as an internal standard. Each mixture was diluted t o volume with the mobile phase. Procedure. Twenty microliters of the reference standard solution was injected into the liquid chromatograph to determine the retention volumes of the compounds. Duplicate standard and sample solutions were chromatographed and the respective responses were determined using peak height measurements. Calculation, Response ratios of both the sample and reference standard were calculated. Per cent panthenol in the sample was obtained by dividing the response ratio of the sample by that of the standard and multiplying by 100. RESULTS AND DISCUSSION Traditionally, the chemical analysis of panthenol has involved the hydrolysis of the amide group followed by reaction with reagents that would enable spectrophotometric measurement. In this procedure for the HPLC determination of panthenol in bulk, premix and liquid multivitamin preparations, aminopropanol, which is formed by the hydrolysis of panthenol, is reacted with fluorescamine, a reagent specific for primary amines (12). The aminopropanol-fluorescamine product is injected into the chromatograph and measured by using either a spectrofluorometer with the excitation wavelength set at 390 nm and the emission wavelength set at 475-490 nm or by measuring the absorbance a t 390 nm with a variable wavelength detector. Absorbance measurements 0 1980 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 52, NO. 8, JULY 1980
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Table I. Analysis of Panthenol-Containing Samples no.
sample
1 2
d-panthenol, bulk dl-panthenol, bulk 33%d-panthenol ViPenta multivitamin drops Berocca-C 2-mL vials Berocca-C 20-mL vials
3 4 5 6
a Nonaqueous titration with perchloric acid. Microbiological assay.
theoretical
HF’LC, found
assay, other methods
35% 1 5 mg/0.6 mL 24 mg/2 mL 240 mg/20 mL
99.6% 100.5% 36.1% 14.8 mg/0.6 mL 23.5 mg/2 mL 237.6 mg/20 mL
101.4%‘ 34.5%b 14.1 mg/0.6 mLC 23.1 mg/2 m L C 229.0 mg/20 mLC
100.8%a
Colorimetric determination with 1,2-naphthoquinone-4-sulfonate.
/
Flgure 1.
Dependence of panthenol hydrolysis on acid strength
are adequate for all but trace analysis, where fluorometric detection must be used. T h e hydrolysis parameters involving acid strength and reaction time were studied by monitoring the relative absorbance of the hydrolysis product formed after reaction with fluorescamine. Identical concentrations of panthenol were hydrolyzed with different acid strengths ranging from 0.1 to 2.0 N HC1 for 30 min in a constant temperature water bath maintained a t 85 f 2 “C. T h e data in Figure 1 show that complete hydrolysis was obtained using 0.5 N HC1. Higher acid concentrations did not produce any increase in absorbance due to additional reaction product formation. T h e aminopropanol-fluorescamine derivative was chromatographed on a reversed-phase column using a mixture of borate buffer (pH 8) and methanol as the eluant. Despite the high mobile phase pH, no sign of column deterioration was observed for the 4-month time that the column was used. A typical chromatogram is shown in Figure 2 with t-aminocaproic acid as internal standard. Since the derivatization reaction is very dependent on the pH of the solution, an internal standard that reacts similarly to the sample was chosen in order to be able to standardize the conditions. In a multivitamin preparation where a large amount of riboflavin is present and may not be fully resolved chromatographically from e-aminocaproic acid (which elutes immediately after riboflavin), aminoethanol can be used as an internal standard. T h e multivitamin samples that were analyzed in this study did not require the use of aminoethanol as the internal standard. A linear relationship was observed for the plot of detector response vs. the amount of aminopropanol-fluorescamine derivative equivalent to between 0.4 and 1.8 pg of panthenol injected. Table I shows a variety of samples analyzed for panthenol content using this procedure. The results obtained by HPLC were in close agreement with the theoretical amount of substance claimed in the sample and also were found comparable with those obtained using different assay procedures employed for the particular preparation. Data obtained from six in-
1 , 32
~:
:
~
:t-H
16 B 24 R E T E N T I O N VOLUME. ML
0
Flgure 2. Chromatogram for the determination of panthenol after reaction with fluorescamine using e-aminocaproic acid as internal standard
i Chromatograms for the determination of free-aminopropanol in panthenol. (A) Standard, (B) sample Figure 3.
jections each of three different panthenol-containing samples had a relative standard deviation of 2-370.
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Anal. Chem. 1980, 52, 1370-1371
Panthenol is available in various grades which have specific uses in pharmaceutical preparations; the grade is determined by the amount of free aminopropanol (precursor to panthenol) present in the sample. An existing method for free aminopropanol employs potentiometric titration (11) in which very large amounts of sample are required in order to obtain an accurate result. This method may be used to determine the free aminopropanol content of samples if the hydrolysis step of the procedure is omitted. Figure 3 demonstrates the applicability to the determintion of free aminopropanol at a level of 0.1% on a 4-mg/mL sample solution. T h e method discussed in this report is specific and rapid for the determination of panthenol and aminopropanol, and can be used to monitor trace levels of these compounds. The procedure may also be applied to the analysis of other amine-containing compounds.
LITERATURE CITED (1) Rogers, G. G.; Campbell, J . A. Anal. Cbem. 1960, 32, 1662. (2) Myszkowska, K.; Tautt, J.; Tuszynska, S.; Wozniak, W. Acta Polon. Pbarm. 1964, 21, 84. (3) Bird. 0.D.; McCready, L. Anal. Cbem. 1958, 30, 2045. (4) Matta, G.; Lopez, E. S . Rev. Farm. 1966, 16, 452. (5) Painier, R. G.; Close, J. A. J . Pharm. Sci. 1964, 53, 108. (6) Wollish, E. G.; Schrnall, M. Anal. Chem. 1957, 29, 1509. (7) Wollish, E. G.;Schrnall, M. Anal. Chem. 1950, 22, 1033. (8) Vacheck, J . Pharmazie 1966, 21, 222. (9) Zappala. A. F.; Simpson, C. A. J . Pbarm. Sci. 1981, 50, 845. (10) Bergmann, F. Anal. Cbem. 1952, 24, 1367. (1 1) Pifer, C. W. Unpublished work, “The Chemical Assay of Bulk Panthenol”; Hoffmann-La Roche Inc.: Nutley, N.J., 1956. (12) Weigele, M.; De Bernardo, S.; Tengi, J.; Leimgruber, W. J . Am. Ctwm. SOC. 1972, 94, 5927.
RECEIVED for review October 10, 1979. Accepted March 27, 1980.
Nomogram for Adjusting Mobile Phase Composition in Reverse Phase High Pressure Liquid Chromatography James L. Meek Laboratory of Preclinical Pharmacology, National Institute of Mental Health, Saint Elizabeth’s Hospital, Washington,
One of the first steps in the designing of an assay of a compound by reverse phase high pressure liquid chromatography (HPLC) is to find the mobile phase composition that produces an optimal elution time for the compound. If an initial trial shows that the compound elutes either too close to the time of the void volume or too slowly for convenience, a series of trials is required with decreased or increased, respectively, concentrations of organic solvent until the elution time is in the desired range. If the relationship between retention and concentration of organic solvent were known, it would be possible after the initial trial to calculate just how much to change the percent of organic solvent in order to achieve an optimal retention time. There have been a few studies ( I , 2 ) involving mobile phase composition and retention, but they were limited in the range of polarity of compounds or range of solvent concentrations studied. This paper will show that a simple relationship can be used to describe retention on an octadecylsilyl column vs. mobile phase composition for compounds with a wide range of polarity (catecholamines to anthracene), and a wide range of mobile phase conditions (2.5-80% acetonitrile or methanol in HzO).
EXPERIMENTAL The HPLC apparatus consisted of a Rheodyne sample valve, an Altex model llOA pump, a 25 cm X 4 mm BioRad octadecylsilyl column (10-pmparticle size), Altex-Hitachi variable wavelength spectrometer, and Houston Instruments recorder. Mobile phases consisting of 2 5 8 0 % (v/v) organic solvent, 0.1% H3P04(to adjust the pH to 2.1 (3))and 0.1 M NaC104 (to prevent tailing of the basic compounds) were pumped at room temperature at 1.5 mL/min through the column. The measure of retention used was k’, defined as ( t , - t o ) / t o where t, is the retention time of the compound, and t ois the elution time of an unretained compound. For an unretained compound, NaN03 was chosen. For this study, mobile phase composition vs. k’data were excluded when the observed k’was less than 0.4 or greater than 30. Regression lines were fitted using the unweighted least squares method. 0003-2700/80/0352-1370$01.00/0
D.C. 20032
RESULTS AND DISCUSSION Retention was measured for 10 compounds listed in Figure 1 including neutrals, acids, and bases a t mobile phase concentrations of acetonitrile in water or methanol ranging from 2.5 to 80% (v/v). When log k’was plotted vs. log % acetonitrile (Figure l),essentially straight lines were obtained for each compound. The correlation coefficient r for the compounds ranged between 0.992 and 0.999. However, the slope and y intercept differed for each compound (increasing with increasing lipophilicity). Qualitatively similar data (not shown) were obtained with methanol, the methanol giving longer retention times than acetonitrile for each compound a t any given percent solvent composition. Since log k’ was approximately a linear function of log 70 organic solvent in this range of concentrations, these two parameters were used as the scales for the construction of a nomogram (Figure 2) ( 4 ) . For each compound, lines were drawn connecting each mobile phase composition tested with its observed k’. A point was placed at the intersection of these lines which represents for that compound the relationship between k’and 70 organic solvent. The points for these 10 compounds (Figure 2) lie near a straight line regardless of the compounds’ polarity for both the organic solvents tested. The actual location of a point along that line depends on the polarity of both the solute and the organic solvent, but neither quantity needs to be known to use the nomogram. T o use the nomogram (Figure 2), connect with a straightedge the observed k’ and known mobile phase concentration and note the intersection with the center line. Rotating the straightedge around this point will show what mobile phase conditions would be needed to achieve a given k’ for that compound. As an example (dotted lines), suppose that a k’ of 3 was desired for a compound. In an initial trial a t 10% acetonitrile, the observed k‘was 10. After connecting these points, rotating the straightedge around the point of intersection with the center line reveals that a mobile phase of approximately 20% acetonitrile should be tried to achieve a Q 1980 American Chemical Society
