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ANALYTICAL CHEMISTRY, VOL. 50, NO. 4, APRIL 1978
Determination of Benzodiazepine Anticonvulsants in Plasma by High-Performance Liquid Chromatography Robert J. Perchalski" and B. J. Wilder Research and Neurology Services, Veterans Administration Hospital, and Department of Neurology, College of Medicine, University of Florida, Gainesville, Florida 326 70
A sensitive, specific high-pressure liquid chromatographic procedure for determination of the major benzodiazepine anticonvulsants is described. 4,5-Dihydrodiazepam hydrochloride is easily synthesized and used as internal standard. After a single extraction from alkaline plasma, diazepam, nordiazepam, and clonazepam can be quantitated to a lower limit of 5-10 nglsample. Relative standard deviations for daily and long-term reproducibility studies were 4 % and equal to or less than 6 %, respectively. Preliminary studies indicate that the method can easily be adapted to allow analysis of other benzodiazepines.
T h e pharmacokinetics, metabolism, and possible mechanisms of action of a number of the more significant benzodiazepines have been discussed in detail ( I ) . Two extensive reviews (2, 3 ) published in 1974 indicated that the most popular method of analysis for these drugs was gas-liquid chromatography with electron-capture detection. De Silva et al. ( 4 ) recently published a comprehensive, optimized study of this methodology in which procedures were given for analysis of biological samples containing any single benzodiazepine and its metabolites. Although procedures were optimized for specific groups of drugs, t h e two primary anticonvulsants, diazepam and clonazepam, required different methods of protein precipitation and postextraction handling. This inconvenience prompted us to investigate high-performance liquid chromatography (HPLC) as an alternative method for monitoring these benzodiazepines in the clinical and toxicological situation. Scott and Bommer ( 5 ) described a n HPLC method for analysis of some benzodiazepines and applied it to analysis of animal urine. The chromatography was limited by the state of the art a t that time, but the authors showed the potential of liquid chromatography for separation, identification, and quantitation of benzodiazepines in metabolite studies. More recently, Bugge (6) and Vree e t al. (7) reported analyses of diazepam and nordiazepam and of flunitrazepam, respectively. The mobile phase employed by Bugge is relatively nonspecific a n d barely gives baseline resolution of diazepam and nordiazepam. The procedure is long, and no internal standard is used. Vree and co-workers use diazepam as a n internal standard, so this procedure is not immediately applicable to routine analysis of human samples, since diazepam is a commonly prescribed drug. Both methods employ UV detection at 230 nm, which at the present time requires a variable wavelength detector. T h e expense of this instrument precludes its use in a dedicated routine application. Harzer and Barchet (8) reported on conditions for analyzing several benzodiazepines and their respective benzophenones, using a reverse phase system. In their analysis of whole blood, 5-10 m L of sample were used. Limits of detection were not given for drugs carried through t h e extraction. In this study, we report a rapid (single extraction), sensitive, and selective method, incorporating a n easily synthesized internal standard and UV detection a t 254 nm, for the determination of diazepam, clonazepam, and nordiazepam in 0003-2700/78/0350-0554$01 .OO/O
plasma or whole blood. Some other applications that require little or no modification of the technique are also suggested.
EXPERIMENTAL Apparatus. The liquid chromatograph consisted of an ISCO Model 384 gradient pump with a Model UA-5 absorbance monitor (Instrumentation Specialties Company, Lincoln, Neb.). The optical unit was set up to observe the difference between the mobile phase entering the column and that flowing out of the column at a wavelength of 254 nm. The column was 250 mm X 4.6 mm i.d., packed with a 5-wm totally porous silica gel (Partisil-5, Reeve Angel, Liquid Chromatography Division, Clifton, N.J.). Samples were injected with a six-port sampling valve having a 20-pL sample loop (Valco Instruments Company, Houston, Tex.j. Reagents. All chemicals were analytical reagent grade. Clonazepam and diazepam (Applied Science Laboratories, Inc., State College, Pa.) were used as received. Metabolites of clonazepam were made by catalytic hydrogenation of the parent drug over 5% palladium on carbon in ethanol and by acetylation of the resulting 7-aminoclonazepam with acetic anhydride to obtain 7-acetamidoclonazepam. The internal standard, 4,s-dihydrodiazepam, was made by catalytic hydrogenation of diazepam over platinum oxide in glacial acetic acid according to the method of Sternbach and Reeder (9). Diazepam (100 mg) was dissolved in 1 mL of glacial acetic acid in a 5-mL Reacti-Vial closed with a Tuf-Bond disk (Teflon bonded to silicone) (Pierce Chemical Company, Rockford, Ill.). Two 1.5-mm (1/16-inch)diameter holes were bored through the disk and fitted with 1.5-mm o.d. X 0.3-mm i.d. Teflon tubing to allow introduction (tube extends to bottom of vial) and venting of hydrogen gas. Platinum oxide (10 mg) was added, the vial closed, and hydrogen gas bubbled through the solution for 30 min. A sample of the reaction mix taken at that time, evaporated under vacuum, dissolved in mobile phase, and analyzed by HPLC with UV detection a t 254 nm showed 96% conversion of diazepam to the 4,5-dihydro derivative, 2% unreacted starting material, and two minor products which eluted after 4,5-dihydrodiazepam. The entire solution was centrifuged to separate out the platinum black, and the supernatant uas evaporated to a light brown oil under vacuum. Aqueous 1 M HC1 (2 mL) was added to the residue, and the material was allowed to digest in an 85 O C water bath for 30 min. During that time, the oily residue was converted to an off-white precipitate in a yellow solution. Addition of 1.5-2 mL of methanol to the hot solution was sufficient to just dissolve the precipitate, and the hydrochloride salt crystallized as light yellow needles. A second recrystallization from fresh 1M HC1:methanol (1:lj also gave light yellow needles. The free amine was extremely difficult to recrystallize, and all samples contained a small amount of diazepam impurity. The salt decomposed between 180 and 250 "C. Analysis of part of the residue by dissolution in mobile phase and injection into the liquid chromatograph indicated loss of HC1 to give the free secondary amine and partial reconversion to diazepam. Structures of the clonazepam metabolites and 4,5-dihydrodiazepam were verified by mass spectrometry after purification by semipreparative HPLC. The mobile phase used for quantitation of diazepam, clonazepam, and nordiazepam was cyc1opentane:chloroform:acetonitri1e:rnethanol (29:55.5:15.0:0.5). Flow rate was 60 mL/h at a pressure of 6.67 MPa (967 psi). The internal standard stock solution was made by dissolving about 1 mg of 4,s-dihydrodiazepam hydrochloride (0.887 g base/g salt) in 1 M HC1:methanol (1:9) and diluting to 10 mL. The working internal standard solution was made by adding 0.300 mL 1978 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 50, NO. 4, APRIL 1978 I
2
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mln
Figure 2. Chromatogram of drug standards run under same conditions 1, Peaks represent diazepam ( l ) , 4,5-dihydrodiazepam (2), clonazepam (3),nitrazepam (4), nordiazepam (5), caffeine (6), carbamazepine (7), demoxepam (8),7-chloro-4,5-epoxy-2-methylamino-5-phenyi-3H-l,4-benzodiazepine (tentative) (9), 7-aminoclonazepam (10), chlordiazepoxide (1l), and 7-acetamidoclonazepam (12)
as in Figure
6
,
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min 0
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Figure 1. Extractions of drug-free plasma (A) and drug-free plasma spiked with diazepam ( l ) , clonazepam (3),and nordiazepam (5) at concentrations of 172 ng/mL, 53 ng/mL, and 461 ng/mL, respectively. 4,5-Dihydrodiazepam (2) was added as in Procedure A small amount of caffeine (6) was present. Column: 250 m m X 4.6 mm Partisil (5 pm). Mobile phase: cyclopentane:chloroform:acetonitrile:methanol (29:55.5:15.0:0.5) at 60 mL/h
of internal standard stock solution to 50 mL of 0.1 M ascorbic acid:methanol (9:l). The stock drug standard was made by dissolving diazepam, clonazepam. and nordiazepam (3 mg, 1mg, and 9 mg, respectively) in methanol and diluting to 10 mL. The working drug standard was made by adding 0.300 mL of the stock solution to methanol and diluting to 20 mL with methanol. All standards were stored in silanized amber bottles with Teflon-lined screw caps. Methanolic standards were refrigerated. Plasma standards were made by evaporating 5-100 pL of the methanolic standard in an extraction tube, adding 1.00mL of drug-free plasma to the residue, and extracting. Procedure. To 1.00 mL of plasma in a 16- X 125-mm culture tube with Teflon-lined screw cap, add 0.50 mL of the working internal standard solution, 0.5 mL of glycine buffer (2.0 mol/L, pH 10.51 and 5 mL of benzene:dichloromethane (9:l). After shaking the mixture for 10 min and centrifuging, transfer the organic laver to a 5-mL Reacti-Vial, and evaporate to dryness at ,i"C t? under a stream of nitrogen. Dissolve the residue immediately in 70 PI, of mobile phase, and within 3 days inject approximately one third of the extract (20 pL) into the liquid chromatograph. Quantitate by peak height ratio.
RESULTS AND DISCUSSION Extraction. Various buffers and extracting solvents were evaluated to optimize recovery of all drugs and at the same time keep interferences to a minimum. Concentrated buffers tended to minimize interferences, and the 2 M glycine buffer (pH 10.5) used by Wad and Hanifl (10) for the TLC determinations of diazepam and its metabolites was superior to similar phosphate and borate buffers. Diazepam and nordiazepam are easily extracted by nonpolar solvents (Le., cyclopentane); however, concurrent extraction of clonazepam requires a solvent of higher strength. The benzene:dichloromethane (9:l) solvent of De Silva e t al. ( 4 ) gave good recovery of all drugs without introducing significant interferences. Stability. Peak height ratios of extracted drugs are constant for a t least 3 days if the residue from extraction is dissolved in mobile phase shortly after evaporation of the solvent. A decrease in peak height of the internal standard was repeatedly observed if the residue was allowed to remain
0
5
IO min
Figure 3. Extraction of drug-free plasma spiked with 44.1 ng/mL of nitrazepam (4). Internal standard (2) was added as in the procedure. Chromatographic conditions were as in Figure 1
dry overnight. I t was not necessary to silanize or to use any special cleaning procedures on the extraction tubes or Mini-vials; however, the methanolic standards were more stable if stored in silanized bottles. I t is possible that the benzene:acetone:methanol (80:15:5) solvent used by De Silva et al. ( 4 ) for making up standards may eliminate the need for silanizing storage vessels. Reproducibility. Short-term reproducibility of the method was determined by extracting ten replicates of a drug-free p!asma sample, spiked with diazepam, clonazepam, and nordiazepam at concentrations of 255 ng/mL, 109 ng/mL, and 780 ng/mL, respectively. Relative standard deviations were 4% for all three drugs. Long-term reproducibility was evaluated over a 30-day period (nine determinations) a t two different concentrations of each drug. One set of samples contained 98 ng/mL, 42 ng/mL, and 300 ng/mL of diazepam, clonazepam, and nordiazepam, respectively. The other set contained 294 ng/mL, 126 ng/mL, and 900 ng,'mL, respectively. Relative standard deviations of the set containing low levels of the drugs were 4 % , 6 7 0 , and 670 for diazepam, clonazepam, and nordiazepam, respectively; and 4%, 5 7 0 , and 3.3%, respectively, for the set containing high levels. The range of slopes of analytical curves run a t the beginning and end of this period was within 4% of t,he mean slope for each drug. Recovery. Recovery of each drug was evaluated by calculating concentrations of drugs carried through the extraction by using analytical curves derived from samples to which various concentrations of the three drugs were added after transfer of the organic phase. Equal quantities of the internal
556
ANALYTICAL CHEMISTRY, VOL. 50, NO. 4,APRIL 1978
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Figure 4. Extraction of 2 mL plasma from a patient taking 13 mg/day of clonazepam. Chromatogram shows clonazepam (3), caffeine (6), 7-aminoclonazepam (1O), and expected position of 7-acetamidoclonazepam (12). Column: 250 mm X 4.6 mm Partisil PAC (alkylnitrileFl0 pm. Mobile phase: ch1oroform:acetonitrile:methanol(60:39: 1) at 60 mL/h
0
5
10
15 min
Figure 5. Extraction of 1 mL whole blood from medical examiner subject suspected of taking chlordiazepoxide. Peaks are nordiazepam (5), caffeine (6),demoxepam (8),7-chloro-4,5-epoxy-2-methylamino-5phenyl-3H-l,4-benzodiazepine(tentative) (9), and chlordiazepoxide (1 1). Chromatographic conditions were the same as those in Figure 4.
standard (as the free base) were added to all tubes after transfer of the organic phase. Four replicates, spiked with 138 ng/mL, 42.4 ng/mL, and 268 ng/mL of diazepam, clonazepam, and nordiazepam, respectively, were extracted. Recoveries were 92.9 f 3.3%, 89.6 f 5.2%, and 93.6 f 3.870, respectively. Lower limits of quantitation are 5 ng/mL for diazepam and 10 ng/mL for clonazepam and nordiazepam. The lower limit of detection for clonazepam is 3.1 ng injected with detector noise equal to 1.0 x absorbance unit. Therapeutic concentrations are 200-500 ng/mL, 600-1500 ng/mL, and 40-100 ng/mL for diazepam, nordiazepam, and clonazepam, respectively. Interferences. The chromatograms in Figure 1 show extraction of drug-free plasma (A) and drug-free plasma spiked with 172 ng/mL, 53 ng/mL, and 461 ng/mL of diazepam, clonazepam, and nordiazepam, respectively (B). This procedure has been in use for the past 5 months, and plasma from patients on common anticonvulsant medications contains no interfering peaks. In some plasma, including some drug-free plasma, a large peak is present which elutes immediately before diazepam. Although it does not interfere, it may be mistakenly identified as diazepam in samples that do not contain this drug. A major metabolite of carbamazepine, probably the epoxide, elutes just before nordiazepam. Again, this peak does not interfere with clonazepam or nordiazepam. An interference t h a t has been noted in only one medical
examiner specimen (not from an epileptic patient) has been identified by mass spectral analysis as phenacetin. This compound elutes a t the same time as 4,5-dihydrodiazepam. The short half-life of this drug (1-3 h), and the availability of alternative analgesic medication should make this a minor problem for epileptic patients on benzodiazepine therapy. If the drug must be used, it should not be taken for a t least 24 h before sampling for benzodiazepine analysis. During the initial development of this method, a reverse phase system was also evaluated. Carbamazepine could not be resolved in a reasonable time from clonazepam with water:methanol or water:acetonitrile mobile phases. Other Applications. Numerous other possible applications of this basic method have been indicated, primarily during analysis and identification of unknown compounds present in medical examiner samples. Figure 2 shows the elution pattern of the drugs of interest, along with the internal standard and some related compounds that are easily extracted by this method. This figure was included only to show the chromatographic relationship of related and coextractable substances, and not to suggest that these conditions provide an optimized assay for all components. Caffeine and carbamazepine were included because the former is found in almost all samples and the latter is a common anticonvulsant. An extraction of a plasma sample spiked with 44.1 ng/mL of nitrazepam is shown in Figure 3. To determine whether this method was applicable to quantitation of the clonazepam metabolites, a 250-mm X 4.6-mm i.d. column packed with a 10-pm alkylnitrile bonded-phase packing (Partisil10/25 PAC, Reeve-Angel) was used with ch1oroform:acetonitrile:methanol (60:39:1) as mobile phase a t 60 mL/h. Figure 4 shows an extraction of 2 mL of plasma from a patient on a continual dose of 13 mg of clonazepam/day. The clonazepam level in this sample was 90.1 ng/mL. The 7-acetamido metabolite was not detected. A medical examiner sample from a person suspected of taking chlordiazepoxide (Librium) was extracted according to the procedure and chromatographed under the conditions used for clonazepam metabolites. The resulting chromatogram is shown in Figure 5 . Chlordiazepoxide was quantitated by comparison of peak areas of extracted standards with that of the unknown and found to be about 600 ng/mL. With inclusion of a suitable internal standard, a simple, rapid analysis of chlordiazepoxide and its principal metabolite, demoxepam, is possible. The component that elutes after demoxepam has been tentatively identified by mass spectral and UV absorption studies as the 4,5-epoxide of chlordiazepoxide. This compound has been reported in chemical studies of chlordiazepoxide ( I I ) , but has never been found as a metabolite, probably because of its easy reconversion to chlordiazepoxide with heat or in dilute acid. The proposed method for diazepam, clonazepam, and nordiazepam is rapid, sensitive, and easily applied to routine therapeutic monitoring of epileptic patients on benzodiazepine therapy. The required instrumentation is less expensive and easier to maintain than the more commonly used electron capture gas chromatograph and is more practical for dedicated clinical analysis than the liquid chromatograph with a variable wavelength detector. The method can be applied directly to the determination of nitrazepam and, with a simple change of column and mobile phase, to the determination of chlordiazepoxide and demoxepam. Manipulation of the mobile phase within these two basic systems should allow analysis of many of the other less commonly used benzodiazepines.
ACKNOWLEDGMENT Thanks are due to J. L. Templeton and B. D. Andresen for mass spectral data and to R. M. Thomas and D. M. Mitchell for technical assistance.
ANALYTICAL CHEMISTRY, VOL. 50, NO. 4, APRIL 1978
LITERATURE CITED (1) S. Garrattini, E. Mussini, and L. 0.Randall, Ed., "The Benzodiazepines",
Raven Press, New York. N.Y., 1973.
(2) J. M. Clifford and W. F. Smyth, Analyst (London), 99, 241 (1974). (3) D. M. Hailey, J . Chromatogr., 98, 527 (1974). (4) J. A. F. De Siba, I. Bekersky,C. V. Buglid, M. A. Brooks, and R. E. Weinfekl, Anal. Chem., 48, 10 (1976). ( 5 ) C. G. Scott and P. Bommer, J , Chromatogr. Sci., 8, 446 (1970). (6) A . Bugge, J . Chromatogr., 128, 111 (1976). (7) T. B. Vree, B. Lenselink, E. van der Kieijn, and G. M. M. Nijhuis, J . Chromatogr., 143, 530 (1977). (8) K . Harzer and R. Barchet. J . Chromatogr., 132, 83 (1977).
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(9) L. H. Sternbach and E. Reeber, J . Org. Chem., 26, 4936 (1961). (10) N. T. Wad and E. J. Hanifi, J . Chromatogr., 143. 214 (1977). ( 1 1 ) L. H. Sternbach, B. A. Koechlin, and E. Reeder, J. Org. Chem., 27, 4671 (1962).
RECEIVED November 15, 1977. Accepted January 12, 1978. This work was supported by the Medical Research Service of the Veterans Administration, the Epilepsy Research Foundation of Florida, Inc., and the College of Pharmacy Mass Spectrometry Facility of the University of Florida.
Microdetermination of Molecular Species of Oligo- and Polyunsaturated Diacylglycerols by Gas Chromatography-Mass Spectrometry of Their fert-Butyl Dimethylsilyl Ethers J. J. Myher, A. Kuksis,' L. Marai, and S. K. F. Yeung Banting and Best Department of Medical Research, University of Toronto, Toronto, Canada
Gas chromatography-mass spectrometry (GCIMS) was used to determine the molecular association and positional distribution of fatty acids in submicrogram quantities of diacylglycerols following their conversion into the fert-butyl dimethylsilyl (1-BDMS) ethers. The abundant M - 57 ion provided the molecular weights for both saturated and unsaturated species. The 1,3 isomers were identified by the M - acyloxymethylene fragments which are absent in the sn-1,2and sn-2,3-diacylglyceroIs. The abundance ratio of the ions due to losses of the acyloxy radical (M - RCOO) from position 1 (or 3) and position 2 indicated the proportions of the reverse isomers, e. g. sn-1-palmiloyl, 2-stearoy1, and sn-1-stearoyl 2-palmiloylglycerols.
T h e early methods of complete determination of the molecular species of diacylglycerols were based on combined application of argentation thin-layer chromatography (AgKO,-TLC), gas-liquid chromatography (GLC), and enzymatic degradation ( I , 2 ) . These techniques, although extensively applied in research ( 3 ) ,are laborious and time consuming, and require more material than conveniently prepared from such natural sources as cell membranes and lipoproteins. T h e potential usefulness of mass spectrometry for a rapid analysis of minute amounts of molecular species of diacylglycerols has been recognized ( 4 , 5 )since the introduction of GC/MS methods to the investigation of the structure of complex glycerolipids, b u t t h e relative unavailability of t h e instruments prevented progress in the methodology or its widespread utilization in assessment of natural mixtures of diacylglycerols. Current GC/MS analyses of diacylglycerols utilize trimethylsilyl (TMS) ethers (6-8), which are unstable and cannot be purified before analysis, or acetates ( 9 - l I ) , which d o not yield sufficiently characteristic spectra for polyunsaturates. T h e present study demonstrates that the t-BDMS ethers of diacylglycerols possess many of the mass spectrometric properties of the TMS ethers along with a prominent M 57 fragment, which can be utilized for accurate measurement of molecular weight. The t-BDMS ethers are stable to moisture and can be isolated from the reaction mixture and purified -
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M5G 1L6
prior t o analysis, as previously shown for the corresponding derivatives of simple alcohols (12: I,?).
EXPERIMENTAL Standards. Synthetic mono- and diacid sn-1,2-tliacq-lglycerols of 16 and 18 carbon fatty acids were purchased from Applied Science Laboratories (State College, Pa.) and Supelco (Bellefonte, Pa.). The reverse isomers of rac-1-palmitoyl 2-stearoyl and rac-1-stearoyl2-palmitoyl glycerols were prepared in the laboratory by Grignmd degradation of rac-1-palmitoyl 2-stearoyl3-palmitoyl glycerol and rac-1-stearoyl 2-palmitoyl 3-stearoylglycerol as previously described (11). The triacylglycerols were prepared by acylation with the acid chlorides of the corresponding 1,3-diacylglycerols. Mono-, di-, tri-, tetra- and hexaenoic sn-1.2-diacylglycerols of natural origin were isolated by argentation TLC of the t-BDMS ethers of sn-1,2-diacylglycerols derived from egg yolk and rat liver phosphatidylcholines by hydrolysis with phospholipase C (14). The AgN0,-TLC fractions contained largely mixtures of palmitoyl and stearoyl oleates, linoleates, arachidonates, and docosahexaenoates, respectively, but dioleoyl, oleoyl linoleoyl, dilinoleoyl, and oleoyl arachidonoyl species were also present. Preparation of 1,2-(2,3)- and 1,3-Dipalmitoyl-sn-glycer01-d,. Glycerol-d, (20 mg) was reacted for 4 h s i t 80 "C with palmitoyl chloride (120 mg) in benzene (0.5 mL) containing a catalytic amount of dry pyridine (30 mg). The mixture was extracted with CHCI3/MeOH (2:1,v / v ) and washed with water. Analysis of the products by GLC (15) indicated the presence of mono-, di-, and tripalmitoyl-glycerols. The 1,2(2,3)-and 1,3dipalmitoyl-sn-glycerols-d, were isolated from the mixture by TLC on borate impregnated silica gel using CHC13/acetone (96:4, v j v ) as the developing solvent (16). The diacylglycerols were converted into their respective t-BDMS ethers and purified by TLC on silica gel H as described below. Preparation of t-BDMS Ethers. The t-BDMS ethers of diacylglycerols were prepared by a modification of the method described by Corey and Venkateswarlu (12)for prostaglandins. A mixture of 0.5 mg diacylglycerol and 150 pL tert-butyldimethylchlorosilane/imidazole reagent (Applied Science Lahoratories) is heated at 80 "C for 20 min. .4fter cooling, the reaction mixture is thoroughly mixed with 5 mL of light petroleum spirit (bp 30-40 "C) and washed three times with 0.5 mL of water. The petroleum extract is then dried over Na2S04and taken to dryness under a stream of nitrogen. No isomerization of mono- or diacylglycerols was observed to take place during derivatization as determined by GLC. The t-BDMS ethers of acylglycerols are suitable for TLC and AgN0,-TLC. The t-BDMS ethers can be stored in petroleum ether at -20 "C for protection of double bonds. D 1978 American Chemical Society
