Application of ion-pair high-performance liquid chromatography to detection of the atypical coproporphyrin isomers II and IV in human faeces

A highly selective and sensitive method has been developed for the detection of small amounts of the atypical isomers II and IV of coproporphyrin in human faeces. This method combines liquid—liquid extraction and solid-phase sampling techniques using talc and C₁₈-modified silica gel as the sorbents. Simultaneous separation of the four coproporphyrin isomers I–IV was achieved by isocratic ion-pair high-performance liquid chromatography. Stool samples of healthy subjects (n = 12) contained 1.1 ± 0.4% (mean ± S.D.) isomer II and 2.2 ± 0.9% isomer IV of total coproporphyrins. A somewhat higher content of isomer II (2.7%) and isomer IV (5.4%) was found in faeces of a patient suffering from porphyria variegata.     

Preparative separation of four isomers of synthetic anisodamine by HPLC and diastereomer crystallization

Anisodamine (654‐1), a well‐known cholinergic antagonist, is marketed as synthetic anisodamine (mixture of four isomers, 654‐2) in China. To preparative resolution and comparison of the bioactivities of the four isomers of synthetic anisodamine, current work explores an economic and effective separation method by using preparative high performance liquid chromatography (HPLC) and diastereomer crystallization. Their absolute configurations were established by single‐crystal X‐ray diffraction and circular dichroism method. The purities of each isomer were more than 95%. Among them, 654‐2‐A2 (6R, 2′S configuration) exhibited better effect on cabachol preconditioned small intestine tension more than 654‐2 and other isomers. The direct separation method without using HPLC was tried as well, which was still on progress. This is the first report of the method for preparative separation of four isomers of synthetic anisodamine which could be used for large‐scale production in industry.     

Method for determining monohydroxybenzo[a]pyrene isomers using column-switching high-performance liquid chromatography

A method for determining monohydroxybenzo[a]pyrene (OHBaP) isomers using column-switching high-performance liquid chromatography with fluorescence detection was developed. Eleven of 12 isomers of OHBaP (all except 6-OHBaP) were separated on an alkylamide-type reversed-phase column and, via column-switching, on a beta-cyclodextrin-bonded silica gel column. The detection limits for the OHBaPs were in the range 0.3-8 pg/injection (S/N=3). By using this method, 1-, 3-, and 9-OHBaPs were identified as major metabolites of benzo[a]pyrene in vitro by human recombinant p450 1A1. The method was used to determine OHBaPs in the urine of a nonsmoker subject. After enzymatic hydrolysis of the conjugated metabolites by beta-glucuronidase/aryl sulfatase, the analytes were selectively adsorbed on blue rayon (a cellulose-supported copper phthalocyanine) from the urine matrix. Methanol as the eluting solvent from the rayon gave the best recoveries of OHBaPs and 1-hydroxypyrene (1-OHP) in the range of 91-103%, which was superior to that of the solid-phase extraction method. 1-OHP, a well-known biomarker of the exposure to polycyclic aromatic hydrocarbons, was simultaneously analyzed. Intra- and interday accuracy values for the determination of 3-OHBaP in 200 ml of urine were 95.5 and 100.9%, and those for 1-OHP were 96.4 and 103.6%, respectively. The intra- and interday precision values were 3.9 and 2.4% for 3-OHBaP and 2.4 and 3.2% for 1-OHP, respectively. In 11 kinds of isomers, only 3-OHBaP was detected in the human urine. Urinary concentration of 3-OHBaP was quantified at 0.5 ng/g creatinine concentration and the 3-OHBaP/1-OHP ratio was approximately 1/130.     

Effect of dietary conjugated linoleic acid and selenized yeast on the concentration of fatty acids and minerals in rats

The main objective of this study was to evaluate the influence of diets enriched in individual conjugated linoleic acid (CLA) isomers, their mixture, and/or selenized yeast (Se-yeast) on the concentration of CLA isomers, long-chain polyunsaturated fatty acids (PUFA) and Se in the heart, muscles and liver of rats. The investigation was performed on 73 female Wistar rats (8 weeks of age, 200 g initial BW). After one week sub-maintenance feeding, rats received diets supplemented with 1% individual CLA isomers or 1 or 2% of a CLA isomers mixture, without or with 1.2 mg Se/kg (as Se-yeast) for 29 days. Feeding diets with 2% CLA isomer mixture reduced feed intake and body weight gain of rats, while addition of trans10,cis12 CLA and Se-yeast resulted in the highest body weight gain. CLA supplementation generally elevated the concentration of CLA isomers in heart and muscles significantly, although cis9,trans11 CLA accumulated preferentially. Regardless of the presence of Se-yeast, the dietary enrichment with CLA isomers caused a reduction in the capacity of A9-desaturase. Addition of Se-yeast to diets with individual CLA isomers or a 1% mixture of CLA isomers elevated the accumulation of CLA isomers in the heart and muscles, whereas all treatments with supplemented CLA and Se-yeast increased the accumulation of Se in rats compared with animals fed the diet containing Se only. Furthermore, CLA isomer supplementation decreased the concentration of PUFA and total fatty acids in the heart and muscles compared with control rats. Moreover, addition of CLA isomers interfered in the conversion of linoleic and linolenic acids to higher metabolites due to competition of CLA isomers for the same enzymes (delta6-, delta5-, delta4-desaturases and elongase).     

Quantitative analysis of histidine and cis and trans isomers of urocanic acid by high-performance liquid chromatography: a new assay method and its application

To elucidate the factors involved in dry skin and the skin damage caused by UV light, it is necessary to analyze small amounts of stratum corneum to determine amino acid contents. A new assay method for this purpose is described. Dabsylated amino acids including histidine and the cis and trans isomers of urocanic acid were analyzed quantitatively by high-performance liquid chromatography (HPLC), using a reversed-phase column. Histidine and the isomers of urocanic acid were separated from 36 other amino acids thought to be present in the extract of stratum corneum. In the presence of the 36 amino acids, standard calibration curves were obtained from 0.25 to 2.5 pmol/μl, for histidine and for both isomers of urocanic acid. The coefficients of variation for the reproducibility of the analysis at 1.0 pmol/μl were 3.8%, 2.9% and 2.5% for the cis and trans isomers of urocanic acid and for histidine, respectively. Amounts of 2 to 50 pmol of cis and trans isomers of urocanic acid and histidine in the stratum corneum were detected. The ratio of the cis to the trans isomer of urocanic acid in sunburned stratum corneum was more than three times that in normal stratum corneum. This method appears to be useful for the determination of small amounts of histidine and of the cis and trans isomers of urocanic acid in the stratum corneum.     

Synthesis of the optical isomers of a new anticholinergic drug, penehyclidine hydrochloride (8018)

A practical diastereoselective synthetic method for 8018 enantiopure isomers is described. The intramolecular asymmetric epoxidation of mono-sulfonate 4 was applied for the execution of the synthesis of the key chiral building block for the first time. The isomers were obtained with 70-76% yields in 99-100% ee.     

Quantitative gas chromatographic method for the analysis of cis-9, trans-11 and trans-10, cis-12 isomers of the conjugated linoleic acid in liver

A quantitative GC method for conjugated linoleic acid (CLA) isomers of physiological significance (cis-9, trans-11 CLA and trans-10, cis-12 CLA) as non-esterified fatty acids (NEFA) or triacylglycerols (TAG) was developed. Furthermore, the effect of the internal standard addition point (sample or fat extract) was studied. Response linearity, recovery and precision assays, detection and quantification limits were determined. Linearity was demonstrated over a range from 0.1 to 10 microg/mL. When CLA isomers were present as NEFA, the recovery significantly decreased (P< or =0.05) from 76% to 27.1% (cis-9, trans-11 CLA) and 28.5% (trans-10, cis-12 CLA) when the standards were added to the fat extract or to the initial tissue, respectively. As an application, liver samples from hamsters fed a diet supplemented with both CLA isomers were analyzed. The CLA isomers in liver samples were detected with reasonable reproducibility.     

Isomerization of stable isotopically labeled elaidic acid to cis and trans monoenes by ruminal microbes

A previous study showed that oleic acid was converted by mixed ruminal microbes to stearic acid and also converted to a multitude of trans octadecenoic acid isomers. This study traced the metabolism of one of these trans C18:1 isomers upon its incubation with mixed ruminal microbes. Unlabeled and labeled (18-[13C]trans-9 C18:1) elaidic acid were each added to four in vitro batch cultures with three cultures inoculated with mixed ruminal bacteria and one uninoculated culture. Samples were taken at 0, 12, 24, and 48 h and analyzed for 13C enrichment in component fatty acids by gas chromatography-mass spectrometry. At 0 h of incubation, enrichment was detected only in elaidic acid. By 48 h of incubation, 13C enrichment was 18% (P < 0.01) for stearic acid, 7% to 30% (P < 0.01) for all trans C18:1 isomers having double bonds between carbons six through 16, and 5% to 10% for cis-9 and cis-11 monoenes. After 48 h, 13C enrichment in the uninoculated cultures was only detected in the added elaidic acid. This study shows trans fatty acids exposed to active ruminal cultures are converted to stearic acid but also undergo enzymic isomerization yielding a multitude of positional and geometric isomers.     

Effect of dietary conjugated linoleic acid and selenized yeast on the concentration of fatty acids and minerals in rats

Abstract

The main objective of this study was to evaluate the influence of diets enriched in individual conjugated linoleic acid (CLA) isomers, their mixture, and/or selenized yeast (Se-yeast) on the concentration of CLA isomers, long-chain polyunsaturated fatty acids (PUFA) and Se in the heart, muscles and liver of rats. The investigation was performed on 73 female Wistar rats (8 weeks of age, 200 g initial BW). After one week sub-maintenance feeding, rats received diets supplemented with 1% individual CLA isomers or 1 or 2% of a CLA isomers mixture, without or with 1.2 mg Se/kg (as Se-yeast) for 29 days. Feeding diets with 2% CLA isomer mixture reduced feed intake and body weight gain of rats, while addition of trans10,cis12 CLA and Se-yeast resulted in the highest body weight gain. CLA supplementation generally elevated the concentration of CLA isomers in heart and muscles significantly, although cis9,trans11 CLA accumulated preferentially. Regardless of the presence of Se-yeast, the dietary enrichment with CLA isomers caused a reduction in the capacity of Δ9-desaturase. Addition of Se-yeast to diets with individual CLA isomers or a 1% mixture of CLA isomers elevated the accumulation of CLA isomers in the heart and muscles, whereas all treatments with supplemented CLA and Se-yeast increased the accumulation of Se in rats compared with animals fed the diet containing Se only. Furthermore, CLA isomer supplementation decreased the concentration of PUFA and total fatty acids in the heart and muscles compared with control rats. Moreover, addition of CLA isomers interfered in the conversion of linoleic and linolenic acids to higher metabolites due to competition of CLA isomers for the same enzymes (Δ6-, Δ5-, Δ4-desaturases and elongase).     

Agonist-dependent generation of lipoxins from rat basophilic leukemia cell (RBL-1)

Incubation of RBL-1 cells in the presence of 15-HPETE and various agonists generated lipoxins and several isomers. Addition of either A23187, fMLP or PMA modulated the number of isomers and amount of lipoxins produced. Administration of A23187 yielded the largest amount of product (5.3 +/- 1.6 micrograms per 10(8) cells) and generated a total of six and three isomers of LXA4 and B4, respectively. This was 2-fold greater than fMLP, which produced a total of two isomers of LXA4 and LXB4. Addition of PMA generated only LXA4 (0.68 +/- 0.26 micrograms). This is similar to the control receiving only 15-HPETE. Biologically derived LXA4 (3 nM) isolated from RBL-1 incubations contracted a rat tail artery preparation to 12% of the maximum induced by phenylephrine (0.125 microM), whereas LXA4 standard (3 nM) elicited 17.6% of the maximum contraction. These results indicate that RBL-1 cells can utilize exogenous 15-HPETE to generate biologically active lipoxins. Further, the yield and isomers of lipoxins can be modified by different agonists.     

Fully oxidized scrambled isomers are essential and predominant folding intermediates of cardiotoxin-III

Scrambled isomers (X-isomers) are fully oxidized, non-native isomers of disulfide proteins. They have been shown to represent important intermediates along the pathway of oxidative folding of numerous disulfide proteins. A simple method to assess whether X-isomers present as folding intermediate is to conduct oxidative folding of fully reduced protein in the alkaline buffer alone without any supplementing thiol catalyst or redox agent. Cardiotoxin-III (CTX-III) contains 60 amino acids and four disulfide bonds. The mechanism of oxidative folding of CTX-III has been systematically characterized here by analysis of the acid trapped folding intermediates. Folding of CTX-III was shown to proceed sequentially through 1-disulfide, 2-disulfide, 3-disulfide and 4-disulfide (scrambled) isomers as folding intermediates to reach the native structure. When folding of CTX-III was performed in the buffer alone, more than 97% of the protein was trapped as 4-disulfide X-isomers, unable to convert to the native structure due to the absence of thiol catalyst. In the presence of thiol catalyst (GSH) or redox agents (GSH/GSSG), the recovery of native CTX-III was 80-85%. These results demonstrate that X-isomers play an essential and predominant role in the oxidative folding of CTX-III.     

Enzymic assays for isomers of 2,6-diaminopimelic acid in walls of Bacillus cereus and Bacillus megaterium.

An enzymic assay for individual isomers (meso-, LL- and DD-) of 2,6-diaminopimelate was developed. The enzyme 2,6-diaminopimelate decarboxylase specifically attacked meso-diaminopimelate and was used to measure this isomer manometrically. The meso- and LL-isomers were measured together manometrically in a coupled assay with diaminopimelate decarboxylase and diaminopimelate epimerase (which converts LL-diaminopimelate into meso-diaminopimelate). The DD-isomer was not attacked by either enzyme and was measured, as residual diaminopimelate after the coupled assay, by a colorimetric method, which was also used to measure total diaminopimelate before enzymic treatments. The coupled enzymes were also used to prepare pure DD-isomer from chemically synthesized diaminopimelate. A mixture of diaminopimelate isomers was present in walls of four strains of Bacillus megaterium [in each about 75% (w/w) meso-, 18% LL- and 7% DD-] and in walls of two strains of Bacillus cereus (about 85% meso-, 8% LL- and 7% DD-). One strain of B. cereus contained at least 95% meso-diaminopimelate, with only traces of LL- and DD-isomers. Peptidoglycan from Escherichia coli was assayed as containing at least 95% meso-isomer. The proportion of isomers in the wall of a strain of B. megaterium remained constant after growth in a variety of different media.     

Quantitative analysis of chiral compounds from unresolved peaks in capillary electrophoresis using multivariate calibration with experimental design and artificial neural networks

Quantitation of optical isomers can be achieved even from incompletely resolved peaks with a multivariate calibration applying a combination of experimental design and artificial neural networks (ANN). Using the proposed approach, method development can be more efficient and analysis time shortened while quantitation of optical isomers with acceptable precision (+/-1-3%) can be achieved.     

Incorporation of dietary cis and trans isomers of octadecenoate in lipid classes of liver and hepatoma.

Groups of rats bearing Morris minimal deviation hepatoma 7288CTC were fed a fat-free diet supplemented with either 0.5% safflower oil (diet A), 15% safflower oil or free acids (diets Band C), or 15% safflower oil or free safflower fatty acids (diet D) for 4 weeks. A group of normal rats was also fed diet D. Triglycerides, cholesteryl esters, phosphatidylcholines, and phosphatidylethanolamines isolated from livers and hepatomas of animals on each diet were analyzed quantitatively for positional isomers in the cis- and trans-octadecenoate fractions. When sufficient samples could be obtained, the cis- and trans-hexadecenoate fractions were also analyzed. Plasma from normal rats on diet D was analyzed in the same manner. The octadecenoate fractions of all hepatoma and liver lipid classes from animals fed diets A, B, and C were greater than 95% the cis isomers. Trans isomers accounted for approximately 15, 30, 50, and 70% of the octadecenoate fractions isolated from liver triglycerides, cholesteryl esters, phosphatidylcholines, and phosphatidylethanolamines, respectively, of animals fed diet D. In contrast, all hepatoma lipid classes from animals on diet D contained the same approximate percentage of trans isomers (15 to 20%). Oleic and vaccenic acids were the major positional cis-octadecenoate isomers of all liver and hepatoma lipid classes from animals fed diets A, B, and C. The ratios of oleic to vaccenic, unaffected by diets A, B, and C, differed for each lipid class in liver, but the ratios were similar for the two hepatoma neutral lipid classes and for the two phospholipid classes. The cis-octadecenoate fractions from all liver and hepatoma lipid classes of animals fed diet D consisted predominantly of the delta9, delta11, and delta12 isomers. The cis delta10 isomer, which was a major isomer of the diet, was almost excluded from liver, hepatoma, and plasma lipids. The positional isomers of the trans-octadecenoate fractions from liver and hepatoma triglycerides and cholesteryl esters exhibited the same approximate distribution as the trans fatty acids of diet D. In contrast, the 10-trans-octadecenoate, like 10-cis-octadecenoate, was almost excluded from the phospholipids of liver and plasma. Unlike liver, the hepatoma phospholipids contained 10-trans-octadecenoate at approximately half the percentage of neutral lipids. Because diet D contained no hexadecenoic fatty acids, the occurrence of trans-hexadecenoate isomers in liver and plasma lipids indicated a chain shortening process. Predominance of the 8-trans-hexadecenoate isomer indicated a preference of the 10-trans-octadecenoate isomer for chain shortening.     

Reduction of linoleic acid inhibition in production of conjugated linoleic acid by Propionibacterium freudenreichii ssp. shermanii

A method for the production of conjugated linoleic acid (CLA) from linoleic acid (LA) using growing cultures of Propionibacterium freudenreichii ssp. shermanii JS was developed. The growth inhibitory effect of LA was eliminated by dispersing it in a sufficient concentration of polyoxyethylene sorbitan monooleate detergent. For the whey permeate medium used, the optimum LA:detergent ratio was 1:15 (w/w). As a result, the cultures tolerated at least 1000 microg x mL(-1) LA, which was converted to CLA with 57%-87% efficiency. The cis-9, trans-11 and trans-9, cis-11 isomers constituted 85%-90% of the CLA produced. The feasibility of the method was demonstrated also in de Man Rogosa-Sharpe (MRS) broth.     

On the role of pentachlorocyclohexene in the metabolism and action of hexachlorocyclohexane. I. Synthesis of beta-pentachlorocyclohexene and its identification as the monodehydrochlorination product of alpha-hexachlorocyclohexane (author's transl)]

It is the aim of a series of investigations to test whether or not beta-pentachloro-1-cyclohexene is an intermediate in the biodegradation of alpha-hexachlorocyclohexane. This paper describes attempts to synthesize this intermediate by chemical methods. 1) Pentachlorocyclohexene was synthesized by partial additive chlorination of chlorobenzene. Combined gas chromatography-mass spectrometry revealed that at least five different isomers of pentachlorocyclohexene had been formed. 2) Treatment of alpha-hexachlorocyclohexane with alkaline buffer (pH 8) produced trichlorobenzenes and, in small yield (4%), a pentachlorocyclohexene. This was isolated and identified as the beta-isomer by melting point (71.8 - 72.6 degrees C, uncorr.), IR- and mass spectrum. Dehydrochlorination of beta-pentachlorocyclohexene produced the trichlorobenzene isomers in a pattern which is characteristic of alpha-hexachlorocyclohexane. The position of the chlorine substituents in the beta-pentachlorocyclohexene molecule as judged from NMR studies is e-aeee. This confirms that it is the monodehydrochlorination product of alpha-hexachlorocyclohexane. The configurations of gamma- and delta-pentachlorocyclohexene, determined for comparison, are e-eeaa and e-eeee, respectively. The kinetics of dehydrochlorination of both alpha-hexachlorocyclohexane and beta-pentachlorocyclohexene in alkaline acetone/water (3 + 2) was studied by means of conductometry. Both reactions are of second order: kappa alpha-HCH 0.0495 [1 times mol- minus 1 times s- minus 1[; kappa beta-PCH 0.905 [1 times mol- minus 1 times s- minus 1] (3.6 degrees C). 3) Dehydrochlorination of alpha-hexachlorocyclohexane in pyridine/xylene (3 + 4) was also studied. An earlier report claiming that gamma-pentachlorocyclohexene (and not the beta isomer) is produced in this medium was confirmed, if the reaction was performed at high temperature (120 - 140 degrees C). Moreover, the ratio of trichlorobenzene isomers formed from alpha-hexachlorocyclohexane shifted to a pattern characteristic of the gamma (or gamma) isomer. However, at temperatures of 90 degrees C or less, beta-pentachlorocyclohexene was the main product. The results strongly suggest that in pyridine/xylene, the same isomer is primarily produced from alpha-hexachlorocyclohexane and is isomerized to the gamma, delta and at least two other isomers of pentachlorocyclohexene before further dehydrochlorination ensues. A simple method for the synthesis of beta-pentachlorocyclohexene is presented.     

Application of a rapid and efficient h.p.l.c. method to measure bilirubin and its conjugates from native bile and in model bile systems. Potential use as a tool for kinetic reactions and as an aid in diagnosis of hepatobiliary disease.

We have developed an extremely rapid and efficient reverse-phase h.p.l.c. method for the measurement of bilirubin and its conjugates in human bile and in model bile systems. Our method involves the use of a Perkin-Elmer 3 mu C18 column and a methanol/sodium acetate/aq. ammonium acetate buffer system. Three isomers of bilirubin diglucuronide (BDG), two isomers of bilirubin monoglucuronide (BMG), three isomers of unconjugated bilirubin (UCB) and minor conjugates containing glucose and xylose were separated in 12 min. Initial quantification of BDG and BMG was based on the use of the ethyl anthranilate azo derivative of bilirubin (AZO UCB); however, the standard curves for BDG, BMG and UCB were similar enough to permit quantification to be later based on the UCB standard curve only, thereby simplifying the quantification process. Routine direct injection of 6 or 10 microliter of crude undiluted or diluted (1:1) bile sample was sufficient for analysis. The method was helpful in diagnosing biliary-tract obstruction in a newborn and a partial deficiency state of bilirubin conjugation (Crigler-Najjar syndrome) in a 10-year-old male. When the method was applied to biles of patients both with and without gallstones, levels of UCB were less than 2% of total pigment, consistent with previous reports. Because of its speed and efficiency, this method has the potential for a broad range of applications including enzymic, kinetic and bile sample analyses.     

Separation and evaluation of the cis and trans isomers of hydroxyprolines: effect of hydrolysis on the epimerization

A procedure has been developed which can detect the hydroxyproline isomers trans-4-hydroxyproline (Hyp), trans-3-hydroxyproline, cis-4-hydroxyproline, and cis-3-hydroxyproline present in hydrolysates of collagens. The method involves hydrolyzing collagen, and reacting the primary amino acids with o-phthaladehyde (OPA) and the hydroxyprolines and proline with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) which combines specifically with secondary amino acids. The proline and hydroxyprolines are then separated by thin-layer chromatography and quantified by using a scanning spectrofluorometer. The method was used to show that both trans-4-L-hydroxyproline and trans-3-L-hydroxyproline were epimerized as a function of hydrolysis time to the cis isomers. An appreciable amount of trans-3-Hyp was degraded. Hydrolysis with 6 N HCl in the presence of 6% trichloroacetic acid gave greater epimerization than the 6 N HCl alone. Alkaline hydrolysis in 0.2 M Ba(OH)2 caused more epimerization of trans-4-Hyp and trans-3-Hyp compared with acid hydrolysis but less degradation, so that alkaline hydrolysis is proposed for the evaluation of trans-3-Hyp, provided that the total of the cis and trans isomers be considered in this case.     

Stereoselective and simultaneous measurement of cis- and trans-isomers of doxepin and N-desmethyldoxepin in plasma or urine by high-performance liquid chromatography

Doxepin is a tricyclic antidepressant marketed as an irrational mixture of cis- and trans-geometric isomers in the ratio of 15:85. A convenient high-performance liquid chromatographic (HPLC) procedure for simultaneous quantitation of geometric isomers of doxepin and N-desmethyldoxepin in plasma and urine is described. The HPLC procedure employed a normal phase system with a silica column and a mobile phase consisting of hexane-methanol-nonylamine (95:5:0.3, v/v/v), a UV detector and nortriptyline as the internal standard. The liquid-liquid extraction solvent was a mixture of n-pentane-isopropanol (95:5, v/v). The limit of quantitation was 1 ng/ml for each isomer. The calibration curves were linear over the ranges 1–200 ng/ml (plasma) and 1–400 ng/ml (urine). In plasma, the accuracy (mean±S.D.) (97.53±1.67%) and precision (3.89±1.65%) data for trans-doxepin were similar to corresponding values for urine, i.e., 97.10±2.40 and 3.82±1.14%. Accuracy and precision data for trans-N-desmethyldoxepin in plasma were 97.57±2.06 and 4.38±3.24%, and in urine were 97.64±3.32 and 5.26±1.83%, respectively. Stability tests under three different conditions of storage indicated no evidence of degradation. The recovery of doxepin was 61–64% from plasma and 63–68% from urine. The method has been applied to analyses of plasma and urine samples from human volunteers and animals dosed with doxepin.