(4) E. Watson, S. Wilk, and J. Roboz. Anal. Biochem., 59, 441 (1974). (5) J. B. Brooks and C. C. Alley, Anal. Chem., 46, 145 (1974). (6) R. V. Smith and S. L. Tsai, J. Chromatogr., 61, 29 (1971). (7) J. B. Brooks, C. C. Alley, and J. A. Liddle, Anal. Chem., 46, 1930

procedure has two disadvantages. One, the amount of derivative is about 10 times less than the amount detected by the procedure in reference 7 because of the limited amount of ethanol. Two, the resolution of the ethyl lactate heptafluorobutyrate peak from the trichloroethyl heptafluorobutyrate peak is extremely poor. A much better method for the EC-GLC determination of lactic acid is presented in a previous paper (7). The trichloroethyl ester of formic acid is also very poorly resolved from the trichloroethyl heptafluorobutyrate. Although the application of this procedure to other types of carboxylic acids presents problems, it has been demonstrated in this paper that it is a simple and sensitive EC-GLC method for detecting many of the short chain acids in spent culture media and in body fluids.

(1974). (8) J. E. Brooks, M. J. Selin, and C. C. Alley, An Electron Capture Gas Chromatography Study of the Acid and Alcohol Products of Clostridium

septicum and Clostridium cbauvoei, manuscript in preparation. J. M. Tedder. Chem. Rev., 55, 787 (1955). F. W. McLafferty and R. S. Gohlke, Anal. Chem., 31, 2076 (1959). E . M. Emery, Anal. Chem., 32, 1495 (1960). R. M. Silverstein and G. C. Bassler, "Spectrometric Identification of Organic Compounds," 2nd ed., John Wiley 8 Sons, Inc., New York, 1967, p 29. (13) F. W . McLafferty. Anal. Chem., 31, 82 (1959). (14) L. V. Holdeman and W. E. C. Moore Anaerobe Laboratory Manual, 2nd ed., VPI Anaerobe Laboratory, Virginia Polytechnic Institute and State University, Blacksburg, Va., 1973, p 87.

(9) (10) (11) (12)

LITERATURE CITED

RECEIVEDfor review August 1, 1975. Accepted November 3, 1975. Use of trade names is for identification only and

(1) F. K. Kawahara. Anal. Chem., 40, 2073 (1968). (2) S. W. Dziedzic, L. M. Bertani, D. D. Clarke, and S. E. Gillow, Anal. Biochem., 47, 592 (1972). (3) S. W. Dziedzic, L. B. Dziedzic, and S. E. Gitlow. J. Lab. Clin. Med., 82, 829 (1973).

does not constitute endorsement by the Public Health Service or by the U S . Department of Health, Education, and Welfare.

Electron Capture Gas Chromatography of Tertiary Amines as Pentafluorobenzyl Carbamates Per Hartvig and Wolfgang Hand1 Department of Analytical Pharmaceutical Chemistry, Universityof Uppsala, Box 574,S-757 23 Uppsala, Sweden

Jorgen Vessman* and Carl Magnus Svahn AB Kabi, Research Department, S- 104 25 Stockholm, Sweden

Tertiary amines related to imipramine are reacted with pentafluorobenzyl chloroformate to the corresponding carbamates. Special attention is paid to the quality of the reagent and its influence on the limit of detection. The excess of the reagent, a strong electrophore, is removed by treating the reaction mixture with alcoholic alkali before the gas chromatographic step. The pentafluorobenzyl carbamates have high stability, are formed easily, and show excellent gas chromatographic properties. Down to 3 pg In an Injected sample can be detected in an electron capture detector. The electron capture response is almost Independent of the detector temperature. Quantitative determination of trimipramine in serum were performed in the range of 5-100 ng/ml. The relative standard deviation was 7 % at the 20 ng/ml level.

The high sensitivity of some pentafluorobenzyl carbamates of tertiary amines in the electron capture detector was demonstrated by Hartvig and Vessman (1, 2 ) . Although their gas chromatographic properties were excellent, it was not possible to fully utilize these derivatives in the nanogram range as the excess of the reagent could not a t that time be easily removed. This paper presents an easy way to purify the reaction mixture making derivatization and analysis of nanogram amounts possible. The analysis of serum samples spiked with trimipramine from 5 to 100 ng/ml is demonstrated. 390

EXPERIMENTAL Apparatus. A Varian 1400 gas chromatograph with flame ionization detector (FID) was used with a 0.9 m X 1.8 m m glass column filled with 3% OV-17 on Gas Chrom Q 80-100 mesh. The column temperature was 250 "C and the flow rate of the carrier gas, nitrogen, was 30 ml/min. Studies in the nanogram range were performed in the same instrument with a 6"Ni electron capture detector operated with a constant potential over the electrodes. The chromatographic setup was as above. Detector temperature was 320 "C corresponding to 290 "C a t the foil. The determination of temperature dependence was done in a Hewlett-Packard 5710 A instrument with a frequency modulated '>{Nielectron capture detector. The glass column (1.2 m X 1.8 mm) was filled with 5% OV-17 on Gas Chrom Q 80-100 mesh and operated a t 280 O C . The flow rate of the carrier gas, argon with 5% methane, was 30 ml/min. An LKB 9000 gas chromatograph mass spectrometer was used with a 1.8 m X 3 mm glass column filled with 7% SE-30. The ionization energy was 70 eV. Chemicals a n d Reagents. Pentafluorobenzyl chloroformate was prepared from pentafluorobenzyl alcohol according to methods for the preparation of chloroformates from phenols (3).This reagent is now available from Pierce Chemical Co., Rockford, 111. Heptane was distilled and the purity was checked by gas chromatography. Other reagents used in this study were of the highest analytical quality and were not purified before use. The tertiary amines used in this study were imipramine, trimipramine, and 2chloroimipramine, all of pharmacopoeial quality. Procedures. A Preparation of Pentafluorobenzyi Carbamate T o 0.2 ml of the tertiary amine in heptane (0.5 mg/ml) in a centrifugation tube are added 20 p1 of pentafluorobenzyl chloroformate

ANALYTICAL CHEMISTRY, VOL. 48, NO. 2, FEBRUARY 1976

RESULTS AND DISCUSSION

II

io

I

Figure 1. Gas chromatogram from a reaction mixture of pentafluorobenzyl carbamates after treatment twice with 5 M sodium hydroxide ( 1 ) Pentafluorobenzyl carbamate of trimipramine, 80 ng in 1 ml of water. (2) Pentafluorobenzyl carbamate of 2-chloroimipramine, 89 ng

and about 10 mg of sodium carbonate, anhydrous. T h e heptane solution contains tetratriacontane, which is used as internal standard in these experiments. The tube is attached to an air condenser and the whole setup is heated a t 105 O C in a dry bath. After the appropriate time, 1 ~1 of the reaction mixture is injected in the gas chromatograph equipped with a flame ionization detector, FID. T h e ratio “formed carbamate” to “internal standard” is calculated. R. S t u d i e s o n t h e Remoual of Excess Pentafluorobenzyl Chlorof o r m a t e . T o 1 ml of heptane is added 0.1 ml of t h e pentafluorobenzyl chloroformate reagent. T h e solution also contains tetradecane which is used as internal standard. T h e content in and the change of reagent composition are studied by gas chromatography with temperature programming technique (start a t 100 O C with 1 5 O / min) and flame ionization detection. C. S t u d i e s o n Electron C a p t u r e Response a n d T e m p e r a t u r e Dependence. T h e determination of electron capture response was performed after preparation of the pentafluorobenzyl carbamate in the milligram range, purification of the reaction mixture with 5 M sodium hydroxide, and dilution with heptane to a concentration which on injection of 5 @I gave a signal three times the background noise level. T h e sensitivity is given as the minimum detectable quantity (MDQ) in mol/sec as outlined by Moffat and Homing ( 4 ) . T h e temperature dependence was determined by injection of t h e pentafluorobenzyl carbamate in a n amount that gave a peak about ten times the MDQ value. T h e results are calculated to give a MDQ value. D . Preparation o f S t a n d a r d Curve f r o m S e r u m . 1) T o 1.0 ml of serum spiked with 5-100 ng of trimipramine in a centrifugation tube are added 100 ng of 2-chloroimipramine in 0.1 M phosphoric acid, 0.5 ml of 5 M sodium hydroxide, water to 10 ml, and 10 ml of heptane. T h e tube is extracted for 30 min and centrifuged, if necessary, twice. 2)The water phase is discarded and the organic phase is extracted for 15 min with 2 ml of 0.2 M phosphoric acid. 3) After centrifugation, the water phase is transferred t o another centrifugation tube, alkalized with 0.3 ml of 5 M sodium hydroxide, and extracted for 10 min with 0.2 ml of heptane. 4 ) T h e heptane phase is transferred to a dry reaction tube. Then 20 M I of pentafluorobenzyl chloroformate and 10 mg of sodium carbonate are added. An air condenser is attached to t h e tube a n d , heated a t 105 “Cfor 60 min. 5 ) After t h e reaction, the mixture is treated with 0.5 ml of 0.5 M potassium hydroxide in 75% (w/w) methanol for 5 min. Then 1 ml of water is added and t h e tube is extracted for another 5 min. 6) Finally, 1-3 111 of the organic phase are injected into the gas chromatograph equipped with the electron capture detector.

The Reagent. In previous studies on pentafluorobenzyl chloroformate no application to nanogram analysis was performed ( I , 2) as disturbances from the reagent prevented the use of the full sensitivity of the derivative in the electron capture detector. In order to eliminate these problems, the purity of the reagent and the removal of excess reagent were investigated. Beside pentafluorobenzyl chloroformate, the reagent contains three other components, which are formed during the synthesis. A freshly synthesized reagent contained pentafluorobenzyl chloride (-2%), pentafluorobenzyl alcohol (1%)and dipentafluorobenzyl carbonate (-1%).On contact with moisture, the reagent was gradually decomposed and pentafluorobenzyl alcohol and dipentafluorobenzyl carbonate were formed. When stored in a cool place in the absence of moisture, the reagent was stable. Removal of Excess Reagent after Pentafluorobenzyl Carbamate Formation. As all of the components in the reagent were very sensitive to electron capture detection, it was necessary to remove them completely from the reaction mixture. The purity of the reagent was of importance but, in addition, pentafluorobenzyl alcohol and dipentafluorobenzyl carbonate were also formed during the conditions for the derivative formation. The chloroformate, the concentration of which had diminished in the reaction mixture, could easily be hydrolyzed by treating the reaction mixture twice with 5 M aqueous alkali for 30 rnin a t room temperature. In this treatment, pentafluorobenzyl alcohol was also removed as it was extracted into the aqueous phase. The dipentafluorobenzyl carbonate was not affected by this treatment and an analysis of nanogram amounts was not possible as can be seen from Figure 1. The broad tailing solvent front is the effect of the carbonate which has a retention time long enough but, most important, such a high sensitivity in the detector that it interfered with the carbamate of trimipramine. The resistance of the carbonate towards hydrolysis could be broken if this reaction was performed in a more lipophilic solution, Le., in alcoholic alkali. I t was found with pure carbonate that the methanol content had to exceed 70% t o remove the carbonate completely. This gave two phases. A t 90% methanol, a single phase system was obtained. With 75% methanol, the hydrolysis was completed in less than 1 min. Below 70%, there was no decrease of the chloroformate after 10 min. The hydroxide concentration most suitable for hydrolysis was 0.5 M , With lower concentrations than 0.1 M , the reaction was not completed after 10 min. The treatment of the derivatization reaction mixture with alcoholic alkali also completely removed the alcohol and the chloroformate. The most volatile of the impurities in the reagent, pentafluorobenzyl chloride, was not affected by this treatment and, therefore, this has to be kept a t a low level from the beginning, especially for low boiling derivatives. The carbamates of some amines have been shown to be stable in moderate alkaline and acidic media ( I , 2 ) . The treatment with aqueous alkali decreased the ratio of the pentafluorobenzyl carbamate of imipramine to the internal standard (a hydrocarbon) with less than 5% (Table I), demonstrating the good stability. After treatment with alcoholic alkali, this ratio decreased; but, by dilution of the methanol-containing aqueous phase with water, the original ratio was found, apparently by improving the partition of the carbamate to the organic phase. Reaction Conditions. The reaction conditions evaluated earlier ( I , 2) were used for the compounds in the im-

ANALYTICAL CHEMISTRY, VOL. 48, NO. 2, FEBRUARY 1976

391

Table I. Recovery of the Pentafluorobenzyl Carbamate of Imipramine after Treatment with Alkalia Ratio t o internal standardb

A

A. After derivatization 0.77 B. After treatment twice with 1 mi of 5 M sodium hydroxide 0.74 C. After treatment with 0.5 ml of 0.5 M potassium hydroxide in 75% (w/w) methanol 0.58 D. After dilution of C. with 1 ml of water 0.74 a Experimental conditions: 0 . 2 ml of heptane containing the pentafluorobenzyl carbamate of imipramine (0.5 mg/ ml) and an internal standard (tetratriacontane). Analysis by gas chromatography with FID. b Means of three determinations.

Table 11. Electron Capture Response of PFB-Carbamates M i n i m u m detectable q u a n t i t y X I O L 6 mol/sec (at d i f f e r e n t d e t e c t i o n temperatures) Varian

i

Hewlett-Packard 5710 A

1400

C o m p o u n d as P F B - c a r b a m a t e 3 2 0 O

Imipramine Trimipramine 2-Chloroimipramine

1.3 1.9 1.4

C

180 " C

1.1

.. .

1.2

320

O C

1.6 1.8 1.6

360 " C

1.8

...

1.8

ipramine series except for the concentration of the reagent. T o diminish the disturbances from the reagent, the concentration was reduced from 16 to 8% (v/v). Thereby t h e reaction time was longer, about 1 hour instead of 30 min. Electron Capture Response and Its Dependence on the Detector Temperature. The electron capture response of different pentafluorobenzyl and pentafluorobenzoyl derivatives is extremely high and enables the detection of picogram amounts of an actual compound (5-8). The response of pentafluorobenzyl carbamates formed from difmol sec-' ferent tertiary amines are in the order of which means that down to 3 pg can be detected ( I , 2). The electron capture response of the pentafluorobenzyl carbamates formed from the tertiary amines in the imipramine series in two instruments are shown in Table TI. The attachment of electrons t o different types of compounds in a n electron capture detector has been divided into three different mechanisms (9). Pentafluorobenzoyl derivatives of amines capture electrons according t o a dissociative mechanism, which means almost nondependence on the detector temperature (7). On the other hand, the pentafluorobenzoyl derivatives of alcohols showed their highest response at a low detector temperature (7). As the detector temperature can be used selectively by depressing the response of extraneous material in the chromatogram and with the above discussion in mind on pentafluorobenzyl derivatives, it was of interest to study the temperature dependence of pentafluorobenzyl carbamates over a wide temperature range. This was done in a detector working in the frequency modulated type. The response values a t three different detector temperatures are shown in Table 11. The temperature dependences are very small, indicating a dissociative mechanism. In the Varian 1400 instrument, working with a constant potential over the electrodes, responses of the same order were obtained. The temperature dependence of some other pentafluorobenzyl carbamates in this instrument over a 392

I

1

13

10

5

0

Figure 2. Gas chromatograms from a reaction mixture of pentafluorobenzyl carbamates after treatment with 0 5 M alcoholic potassium hydroxide (1) Pentafluorobenzyl carbamate of inmipramme, 20 ng (2) Pentafluorobenzyl carbamate of 2-chloroimipramine, 90 ng (A) Sample 1 ml of water (B) Sample 1 ml of serum

rather narrow temperature range was previously reported to be low ( I ) . Application to Quantitative Determination of Trimipramine in Serum. Imipramine has earlier been demethylated and derivatized in a two-step reaction leading t o the trifluoroacetyl derivative (IO). The formation of the pentafluorobenzyl carbamate was reported previously (2). None of these procedures were applied to nanogram amounts. With the present procedure to remove the electrophore ,disturbances with alcoholic alkali, it was possible to analyze serum samples spiked with trimipramine in the range of 5-100 ng. 2-Chloro-imipramine was used as an internal standard. As seen from Figure 2A, no disturbances were seen in the chromatogram where an aqueous sample containing 20 ng/ml was analyzed.

ANALYTICAL CHEMISTRY, VOL. 48, NO. 2, FEBRUARY 1976

mate reaction. Preliminary experiments show that this can be done by acylation.

In the analysis of serum samples, the heptane extraction was followed by a re-extraction to get rid of some disturbances from serum. Figure 2B shows a chromatogram from a serum sample with 20 ng of trimipramine per ml. Rectilinear standard curves were obtained in the range of 0-100 ng of trimipramine. The lowest amount that could be determined with the present method was 5 ng in l ml of serum. The relative standard deviation a t the 80- and 20-ng level was 6 and 7% respectively. One important metabolite from the tertiary amines in the imipramine series is the N-demethylated one. Secondary amines form together with chloroformates the same carbamates as tertiary amines do. Therefore, it is essential that the secondary amine be excluded before the chlorofor-

LITERATURE CITED (1) (2) (3) (4) (5)

(6) (7) (8) (9) (10)

P. Hartvig and J. Vessman, Anal. Lett., 7, 223 (1974). P. Hartvig and J. Vessman, J. Chromatog. Sci., 12, 722 (1974). I. C. Raiford and G. 0. Inrnan, J. Am. Chem. SOC.,56, 1586 (1934). A. C. Moffat and E. C. Horning, Anal. Lett, 3, 205 (1970). H. Brotell. H. Ehrsson. and 0. Gyllenhaal, J. Chromatogr., 78, 293 (1973). F. K. Kawahara, Anal. Chem., 40, 1009 (1968). A. Zlatkis and 6. C. Pettitt, Chromatographia, 2, 484 (1969). S. B. Matin and M. Rowland, J. Pharm. Sci., 61, 1235 (1973). W. E. Wentworth and E. Chen, J. Gas Chromatogr., 5, 170 (1967). P. Hartvig and L. Maukonen, Farm. Aikak., 83, 141 (1974).

RECEIVED for review August 8, 1975. Accepted October 22, 1975.

Determination of Lactose Malabsorption by Breath Analysis with Gas Chromatography H. L. Gearhart,'

D. P. Base,* C. A.Smith,*

and

R. D. Morrison3

Oklahoma State University, Stillwater, Okla. 74074

J. D. Welsh and T. K. Smalley Department of Gastroenterology,University of Oklahoma, Health Science Center, Oklahoma City, Okla. 73 790

A method is presented for sampling and analyzing excreted HZ (