Pharmaceutical Chemistry Journal

Vol. 43, No. 8, 2009

STRUCTURE OF CHEMICAL COMPOUNDS, METHODS OF ANALYSIS AND PROCESS CONTROL

GC/MS ANALYSIS OF AQUEOUS ETHANOL SOLUTIONS OF DRUGS. PART 1. MENTHOL AND P-AMINOBENZOIC ACID DERIVATIVES
A. I. Ermakov,1 Yu. Yu. Khomyakov,1 L. P. Soshenko,1 and V. G. Plyushchikov1
Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 43, No. 8, pp. 53 56, August, 2009.
Original article submitted January 21, 2009.

A joint GC/MS analysis of aqueous and aqueous-ethanol solutions of some drugs including menthol, validol, benzocaine, novocaine, and menovazin has been performed using a Varian 3900/2100T (USA) instrument equipped with a Varian Factor Four VF-5ms capillary column. The structures of some impurities that were previously based on theoretical considerations are confirmed. Structural-analytical possibilities of the latest modification of the Varian GC/MS system with an ion-trap source are investigated. It is possible to obtain clear mass spectra even for low-intensity and partly overlapping chromatographic peaks using the given device combination. The results can be used for quality control of drugs based on the investigated compounds. Key words: drugs, menthol, validol, benzocaine, novocaine, menovazin, GC/MS analysis, impurity chemical structure.

GC/MS analysis of aqueous and aqueous-ethanol solutions of compounds I VI (Fig. 1), which are drugs that act on nerve endings, has been performed. Compounds I, IV, and V are ingredients in the drug menovazin. Prepared drugs were purchased at commercial pharmacies and included validol (OAO Farmstandart-Leksredstva, Kursk), menovazin (OAO Tatkhimfarmpreparaty, Kazan), and novocaine (AO Voronezhkhimfarm). First, solutions (1%) in ethanol (70%) of pure compounds I IV and VI were studied. Novocaine (V) was used as an aqueous solution (5%). The solution of menovazin (VI) was studied without dilution and was withdrawn directly from the vial by a microsyringe. It is known that mainly thermal-conductivity detectors, which are the least sensitive detectors compared with other chromatographic detectors, are usually used for chromatographic studies of aqueous and aqueous-ethanol solutions on an ordinary packed chromatographic column. This makes it impossible to analyze reliably and identify in solutions the trace amounts of impurities in the drugs. Therefore, as a rule, drugs are extracted from solutions by organic solvents. This
1

Center for Instrumental Methods and Innovative Analytical Techniques and Materials, Peoples Friendship University, Moscow, Russia.

increases the analysis time. Analytical errors can occur if one or several compounds are incompletely extracted. Menthol is known to exist in eight stereoisomeric forms. The analysis of menthol stereoisomers and their separation in chromatographic columns has been studied for over 30 years [1 4]. Also, mass spectra of menthol and mentholglycol derivatives have been examined [5]. An analytical method using GC for menthol in urine has been developed [6]. GC has been used to determine the quality of novocaine solutions [7]. The authenticity of validol for pharmacopoeic analysis is currently determined by chromatography [8 10]. Our main goal was to study structural-analytical possibilities of a Varian 3900/2100T (USA) GC/MS that was automated and computerized at the most modern level. This addresses the capability of the instrument to record pure mass spectra of very weak chromatographic peaks and the sensitivity of the instrument in general. The instrument was equipped with a database of 365,000 mass spectra of various chemical compounds. The instrument can at the command of the operator output the spectrum and probable structural formula of the chemical compound corresponding to the chromatographic peak. Mass spectra of pure components are studied and their degree of purity is determined beforehand during identifica480
0091-150X/09/4308-0480 2009 Springer Science+Business Media, Inc.

GC/MS Analysis of Aqueous Ethanol Solutions of Drugs

CH3
1

481
a 17.5 15.0 menthol 1 4

R R
2

I, II

25% solution of I and II validol III

12.5
R2

10.0 7.5 5.0 2.5 13.0 35 30 25 1 3 13.5 14.0 b menthol 2

IV, V

Fig. 1. Chemical formulas of studied compounds: R1 = OH, R2 = CH(CH3)2, menthol (I); R1 = OCOCH2CH(CH3)2, R2 = CH(CH3), isovaleric acid menthyl ester (II); 25% solution of I in II validol (III); R1 = NH2, R2 = COOC2H5, benzocaine (IV); R1 = NH2, R2 = COOCH2N(C2H5)2, novocaine (V); 2.5 g (I) + 1 g (V) in ethanol (70%, 100 mL), menovazin (VI).

tion and establishment of the structures of adjuvants in drugs if they consist of several compounds like, for example, menovazin. Although compounds I V have been known for over 100 years and their mass spectra are included in the instrument database, their behavior under electron impact has not to our knowledge been published. Mass spectra of menthol, its menthyl ester, benzocaine, and novocaine that were determined using chromatography agreed fully with spectra in the database. The study of menthol is interesting from the viewpoint of theoretical and practical mass spectrometry (Fig. 2). The menthol molecular-ion peak with m/z 156 was weak, which is characteristic for saturated six-membered rings. The molecular-ion peak of isovaleric acid menthyl ester (II) was missing. One of the strongest peaks corresponded to the ion formed by loss from the molecular ion of neutral isovaleric acid. Its m/z was 138 [M+-HOCOCH2CH(CH3)]+. The intensities of fragment ions in spectra of I and II corresponded to mass numbers with m/z 138 [M+-H2O]+ (menthol), 123 [M+-H2O-CH3]+, 95 [M+-H2O-CHCH3CH3]+, and 81 [M+-OHCHCH3CH3CH3]+ and increased in the order (Irel) 25, 30, 65, 100%, where [M+]+ represents m/z of the molecular ion. Figure 3 shows chromatograms recorded at the maximum scale. The chromatogram in Fig. 3a indicates that the

20 15 10 5 0 12.5 13.0 menthol 10 3 3 2 1 12.5 15.0 17.5 Time, min 20.0 22.5 1 2 4 5 6 7 8 9 2 4

13.5

14.0

14.5 c

5 4

Isovaleric acid menthyl ester 11

12

Fig. 3. Chromatogram of menthol (menthol and impurity peaks shown) (a); part of menovazin chromatogram (menthol and additive peaks shown) (b ); chromatogram of validol isolated from tablets (c). Peaks of isovaleric acid menthyl ester with peaks of additives and the menthol peak also with additive peaks are shown.

% 100

81 297923

75
95 159841

50
83 104165

123 121470

138 99728

25

0 25 50 75 100 125

150 m/z

Fig. 2. Mass spectrum of menthol.

analytical sample of racemic menthol was relatively highly pure. However, four weak chromatographic peaks related to impurities with cyclic six-membered unsaturated structures were clearly recorded. Mass spectra of compounds corresponding to peaks 1 and 2 had molecular ions with m/z 154 [M+-H2]+. The nature of the mass spectra of these compounds (VII VIII) (Fig. 4) were consistent with dehydrated menthol. Compounds corresponding to peaks 3 and 4 had molecular weights m/z 138 [M+-H2O]+. The nature of the mass spectra of these peaks were consistent with compounds IX and X. The chromatograms suggested that the total content of these two components was less than 1%. These same impurities in menthol were detected by chromatography of menovazin, which includes menthol, and had rather high relative intensities, as shown in Fig. 3b. Peak 1

482
CH3 CH3 CH3

A. I. Ermakov et al.

TABLE 1. Chemical Structures of Proposed Components in Validol

Peak No. Structure M+

OH H3C CH3 H3C

OH CH3 H3C CH3

1
IX M m/z 138
CH3
+

VII M m/z 154

CH3
+

VIII M m/z 154

CH3
+

2 3

Unidentified impurity (apparently from tablet filler) Compound XI (Fig. 4)

CH3

136 154

O H3C
OH

CH3

XII 4
CH3

H3C

X + M m/z 138

CH3

H3C

XI + M m/z 136

CH3

H3C

XII + M m/z 150

CH3

154

Fig. 4. Chemical structures of proposed components in menthol.

H3C

O CH3

corresponded to a compound with m/z 154 [M -H2] . The compound of peak 2 had the same mass (VII VIII). Peak 3 corresponded to a compound with m/z 138 [M+-H2O]+. Peak 4 corresponded to a compound with the same molecular mass (138, IX and X). The mass spectra of compounds obtained from peaks 1 2 and 3 4 were significantly different although they had the same [M+]+ values. These facts indicate that these compounds could have the structures shown in Fig. 4. Compounds VII XI in menthol were most probably formed during its synthesis from thymol (XII) by hydrogenation in the presence of a catalyst (Ni, Pt, etc.) [11]. Hence, the menthol studied by us was synthetic. Menthol in nature occurs in essential oil of peppermint as a mixture with the acetic ester. It is obtained by a borate method or freezing from mint essential oil [11]. The picture was more complicated for chromatography of validol (III). We identified using mass spectra [M+]+ peaks of all compounds, the chromatographic peaks of which are shown in Fig. 3c, because they were almost all present in Figs. 2 and 3 with the exception of peak No. 7. The nature of the mass spectrum suggested that this compound was isovaleric aldehyde. The base peaks in the spectrum of this compound were peaks with m/z 86 (molecular ion) and 57, corresponding to a fragment formed by loss of the aldehyde. TABLE 1 lists the proposed chemical structures of the components that corresponded with peaks in the chromatogram of validol and are designated by numbers 1 12. Groups of peaks corresponding to menthol (Nos. 1 6) and the menthyl ester (7 12) were observed. TABLE 1 and Fig. 4 show that components in pure menthol and menthol in validol had the same structures. The structures of the compounds (XII, XIIa, IX, X) were confirmed by spectra of the corresponding compounds from the instrument database. Compounds corresponding to peaks

5 6 7 8 9 10

XIIa Compound IX (Fig. 4) Compound X (Fig. 4) Isovaleric aldehyde Compound X (Fig. 4) Compound XI (Fig. 4)
CH3

138 86 138 136 156

OH H3C CH3

I 11
CH3

156

OH H3C CH3

12

Ia Compound IX (Fig. 4)

138

XII and XIIa (peaks 3 and 4) are obviously geometric isomers. Such isomers are separated in a long capillary column (30 m) and had similar retention times. It is noteworthy that these peaks were not separated in menthol. Compounds IX and X, corresponding to peaks 5 and 6, were structural isomers. This was also confirmed by the identical mass spectra of these compounds from the database. The retention times of these compounds differed rather significantly. This was characteristic of the chromatographic behavior of structural isomers. Therefore, the conclusion can be drawn that the six-membered ring of not only menthol and the menthyl ester

GC/MS Analysis of Aqueous Ethanol Solutions of Drugs

483

but also of the ester molecule in general is destroyed in the chromatographic column. This was indirectly confirmed by the fact that the chromatogram exhibited a peak for isovaleric aldehyde. It is noteworthy that peaks 11 and 12 in the ester were about three times as intense as peaks 5 and 6 in the menthol group. This agreed with menthol and ester concentrations of 1:3 in validol. The FSP [10] addresses the order of elution of validol components as octanol, menthol, and the menthyl ester. Other peaks were not detected. The sequence of validol component elution in the SP [9] was menthene-1, menthene-2, menthene-3, unidentified impurity, neomenthol, menthol, isomenthol, and the menthyl ester of isovaleric acid. It is interesting that peaks corresponding to geometric isomers of neomenthol and isomenthol were detected. The chromatogram was obtained in a 3-m packed column without confirmation of the chemical structure by any physicochemical method. Only the trivial names of these components were given. In our opinion, it is rather complicated to separate geometric isomers of menthol in a 3-m column. GC/MS studies of menovazin detected in its chromatogram p-aminobenzoic acid. Its percent content was less than 0.1%. Clear and sharp peaks of menthol and benzocaine were observed by chromatography of menovazin. Because novocaine is included in the composition of this preparation as the hydrochloride, its chromatographic peak was broad and had an intensity that did not correlate strictly with the amount of injected sample. Only menthol and benzocaine could be determined quantitatively in menovazin without preliminary sample preparation. Preliminary processing of the sample is required to convert novocaine to its base for quantitative determination. The advantages of the Varian 3900/2100T (USA) GC/MS should be mentioned. In contrast with GC/MS with the traditional ion source, the instrument used by us allowed very pure mass spectra to be recorded from weak chromatographic peaks. In our opinion, the compact ion-trap ion-quadrupole analyzer had pracically no memory effect for mass spectra of the preceding peak. Furthermore, this made it possible to obtain clear spectra of partically overlapping chromatographic peaks. This was possible because the scanning frequency of mass spectra in this instrument is very high due to the inertia-free ion source. Such a scanning rate cannot be used in the traditional ion source. This makes this instrument irreplaceable for analyzing trace amounts of impurities in any studied sample.

EXPERIMENTAL CHEMICAL PART Compounds were studied using a Varian FactorFour VF-5ms capillary column of length l = 30 m and diameter d = 0.25 mm on a Varian 3900 (USA) chromatograph. We used a Varian Saturn 2100T mass spectrometer. The ionization method was electron impact with ionization energy 70 eV. Menthol and the menthyl ester were isolated from validol tablets by the literature method [9]. It should be noted that the authenticity of validol and its quantitative composition were determined by GC in a glass column or a stainless steel column with a flame-ionization detector (FID) or thermal-conductivity detector. The chromatography parameters were injector temperature 200C, column temperature (changed with chromatography time according to a program) initial 50C, held for 5 min, linearly increased to 200C at 10C/min. The temperature was held constant for 10 min after reaching the maximum value. The total chromatography time was 30 min. Samples were injected using a Varian CP-8410 auto-injector. The amount of injected sample was 1 mL. The flow ratio was 1:100. The amount of sample fed to the ion source, for example, for menthol, varied in the range 10 3 2 10 3 mg. REFERENCES
1. K. Kogali, K. Aoki, T. Aisaka, et al., J. Jpn. Oil Chem. Soc., 14(3), 129 130 (1965). 2. W. J. Houlihan, Anal. Chem., 34(13), 1846 (1962). 3. M. H. Klowen and R. Heide, Soap Perfum. Cosmet., 35(12), 1082 1083 (1962). 4. N. Uricaru, L. Dimofie, and M. Sterescu, J. Rizescu Rev. Chim. (RSR), 20(2), 68 (1969). 5. M.-G. Ferreti-Aloise, A. Jacot-Guillarmod, and Y.-R. Naves, J. Chem. Acta, 53(2), 201 208 (1970). 6. H. Gleishpach and E. Schanclara, J. Anal. Chem., 252(2/3), 140 143 (1970). 7. N. N. Dement(eva and M. I. Kuleshova, Farmatsiya, 20(4), 17 21 (1971). 8. VFS 42-2394-94, Validol Tablets 0.06 g. 9. FS 42-3392-97 (Kursk KhFZ). 10. FSP 42-0055073801 (OAO Shchelkovskii Vitaminnyi Zavod). 11. G. A. Melent(eva and L. A. Antonova, Pharmaceutical Chemistry [in Russian], Meditsina, Moscow (1985), pp. 287 289.