Determination and Pharmacokinetic Study of Calycosin-7-O-β-d-glucoside in Rat Plasma After Intravenous Administration of Aidi Lyophilizer

Aidi injection is a clinical medicine used in China for the treatment of cancer. Calycosin-7-O-β-d-glucoside is the main effective components of the formulas. In this study, a high performance liquid chromatographic (LC) method was developed to quantify calycosin-7-O-β-d-glucoside in rat plasma using a liquid–liquid extraction and ultraviolet (UV) absorbance detection. LC analysis was performed on a Diamonsil C₁₈ column (200 × 4.6 mm i.d., 5 μm particle size) with isocratic mobile phase consisting of acetonitrile–0.05% phosphoric acid (19.5:80.5, v/v) of a flow rate of 1.0 mL min⁻¹. The linear range was 0.11–17.6 μg mL⁻¹ and the low quantification limit was 0.11 μg mL⁻¹ (S/N = 10). The intra- and inter-day relative standard deviations (RSD) in the measurement of quality control (QC) samples 0.11, 0.22, 1.32 and 8.80 μg mL⁻¹ ranged from 4.1 to 6.3 and 4.3 to 6.2%, respectively. The accuracy was from −6.7 to 4.3% in terms of relative error (RE). Calycosin-7-O-β-d-glucoside was stable in storage at −20 °C for 2 weeks and stable after three freeze–thaw cycles in rat plasma. This method was validated for specificity, accuracy, precision and was successfully applied to pharmacokinetic study of calycosin-7-O-β-d-glucoside in rat plasma after intravenous administration of Aidi lyophilizer.     

Resolution of an intense sweetener mixture by use of a flow injection sensor with on-line solid-phase extraction

An integrated solid-phase spectrophotometry–FIA method is proposed for simultaneous determination of the mixture of saccharin (1,2-benzisothiazol-3(2H)-one-1,1-dioxide; E-954) (SA) and aspartame (N-l-α-aspartyl-l-phenylalanine-1-methyl ester; E-951) (AS). The procedure is based on on-line preconcentration of AS on a C₁₈ silica gel minicolumn and separation from SA, followed by measurement, at λ=210 nm, of the absorbance of SA which is transiently retained on the adsorbent Sephadex G-25 placed in the flow-through cell of a monochannel FIA setup using pH 3.0 orthophosphoric acid–dihydrogen phosphate buffer, 3.75×10^–3 mol L⁻¹, as carrier. Subsequent desorption of AS with methanol enables its determination at λ=205 nm. With a sampling frequency of 10 h⁻¹, the applicable concentration range, the detection limit, and the relative standard deviation were from 1.0 to 200.0 μg mL⁻¹, 0.30 μg mL⁻¹, and 1.0% (80 μg mL⁻¹, n=10), respectively, for SA and from 10.0 to 200.0 μg mL⁻¹, 1.4 μg mL⁻¹, and 1.6% (100 μg mL⁻¹, n=10) for AS. The method was used to determine the amounts of aspartame and saccharin in sweets and drinks. Recovery was always between 99 and 101%. The method enabled satisfactory determination of blends of SA and AS in low-calorie and dietary products and the results were compared with those from an HPLC reference method.     

LC–ESI–MS for Rapid and Sensitive Determination of Aripiprazole in Human Plasma

A rapid, sensitive, and accurate high-performance liquid-chromatographic–mass spectrometric (HPLC–MS) method, with estazolam as internal standard, has been developed and validated for determination of aripiprazole in human plasma. After liquid–liquid extraction the compound was analyzed by HPLC on a C₁₈ column, with acetonitrile—30 mm ammonium acetate containing 0.1% formic acid, 58:42 (v/v), as mobile phase, coupled with electrospray ionization mass spectrometry (ESI-MS). The protonated analyte was quantified by selected-ion recording (SIR) with a quadrupole mass spectrometer in positive-ion mode. Calibration plots were linear over the concentration range 19.9–1119.6 ng mL⁻¹. Intra-day and inter-day precision (CV%) and accuracy (RE%) for quality-control samples (37.3, 124.4, and 622.0 ng mL⁻¹) ranged between 2.5 and 9.0% and between 1.3 and 3.5%, respectively. Extraction recovery of aripiprazole from plasma was in the range 75.8–84.1%. The method enables rapid, sensitive, precise, and accurate measurement of the concentration of aripiprazole in human plasma.     

Separation ofN-carbamoyl aspartate andl-dihydroorotate from serum by high-performance liquid chromatography with fluorimetric detection

Summary A high-performance liquid chromatographic method, with 9-anthryldiazomethane as derivatizing agent, has been developed for the simultaneous determination ofN-carbamoyl aspartate andl-dihydroorotate in serum. Sample preparation for 1 mL serum was by simple liquid-liquid extraction and then derivatization. The compounds were separated on a Luna C18(2) column by use of a gradient prepared from acetonitrile and 10 mM sodium acetate buffer, pH 6.0, and fluorimetric detection was performed at excitation and emission wavelengths of 365 nm and 412 nm, respectively. The response was found to be linearly dependent on concentration between 0.8 and 60 μg mL⁻¹ forl-dihydrooratate and between 0.9 and 90 μg mL⁻¹ forN-carbamoyl aspartate; the mean recovery rates were 50 and 51%, respectively. The limits of detection and quantification were 0.33 μg mL⁻¹ and 0.6 μg mL⁻¹, respectively, forl-dihydroorotate and 0.4 μg mL⁻¹ and 0.7 μg mL⁻¹ forN-carbamoyl aspartate. This method can be used to assess accumulation ofN-carbamoyl aspartate andl-dihydroorotate in body fluids in situations where cellular pyrimidine de novo synthesis is impaired.     

Determination of 5-n-Butyl-4-{4-[2-(1H-tetrazole-5-yl)-1H-pyrrol-1-yl]phenylmethyl}-2,4-dihydro-2-(2,6-dichloridephenyl)-3H-1,2,4-triazol-3-one, a New Angiotensin Type 1 Receptor Antagonist in Rat Plasma by LC-ESI-MS: Application to Pharmacokinetic Studies

A simple and sensitive reversed-phase LC-ESI-MS method to identify and quantitate 5-n-butyl-4-{4-[2-(1H-tetrazole-5-yl)-1H-pyrrol-1-yl]phenylmethyl}-2,4-dihydro-2-(2,6-dichloridephenyl)-3H-1,2,4-triazol-3-one (1b), a new Angiotensin II type 1 receptor antagonist in rat plasma has been developed and validated. Sample preparation used a simple liquid–liquid extraction with ethyl acetate. Separation was achieved by gradient elution on a C₁₈ column. The mobile phase consisted of acetonitrile and water (0.05% triethylamine and 0.05% acetic acid) at a flow rate of 0.2 mL min⁻¹. The detection utilized selected ion monitoring (SIM) in the negative mode at m/z 507.1 and m/z 407.2 for the deprotonated molecular ions of 1b and the internal standard irbesartan, respectively. The lower limit of quantification was reproducible at 5 ng mL⁻¹ with 100 μL of plasma and the good linear was observed in the 5–500 ng mL⁻¹ range. This concentration range corresponded well with the plasma concentrations of 1b in pharmacokinetic studies. Recoveries of 1b in rat plasma were 76.1, 74.6 and 79.0% at 5, 50 and 500 ng mL⁻¹. The RSD of intra-assay and inter-assay variations were all less than 5%. This validated LC-ESI-MS assay is an economic, quick, precise and reliable method for the analysis of 1b in pharmacokinetic studies.     

Simultaneous HPLC Analysis of Olmesartan and Hydrochlorothiazide in Combined Tablets and in vitro Dissolution Studies

A simple, rapid and reproducible HPLC method was developed and validated for the simultaneous determination of olmesartan (OLM) medoxomil and hydrochlorothiazide (HCT) in combined tablets. Chromatography was carried out on a 4.6 mm I.D × 200 mm, 5 μm cyano column with methanol–10 mM phosphoric acid containing 0.1% triethylamine (pH 2.5, 50:50 v/v) at a flow rate of 1.0 mL min⁻¹ and UV detector was set at 260 nm. Valsartan (VAL) was used as internal standard (IS). A linear response was observed in the range of 0.2–6 μg mL⁻¹ (r ² = 0.9998) for OLM and 0.1–4 μg mL⁻¹ (r ² = 0.9999) for HCT, respectively. The method showed good recoveries (99.56% for OLM and 99.48% for HCT) and the relative standard deviation (RSD) values for intra- and inter-day precision were 0.70–1.59 and 0.80–2.00% for OLM and 1.20–1.37 and 1.63–1.93% for HCT, respectively. The developed method was applied successfully for quality control assay of OLM and HCT in combined tablets and in vitro dissolution studies.     

Development of a liquid chromatography-mass spectrometry method for the determination of ursolic acid in rat plasma and tissue: application to the pharmacokinetic and tissue distribution study

A fast and sensitive liquid chromatography–mass spectrometry method was developed for the determination of ursolic acid (UA) in rat plasma and tissues. Glycyrrhetinic acid was used as the internal standard (IS). Chromatographic separation was performed on a 3.5 μm Zorbax SB-C18 column (30 mm × 2.1 mm) with a mobile phase consisting of methanol and aqueous 10 mM ammonium acetate using gradient elution. Quantification was performed by selected ion monitoring with (m/z)⁻ 455 for UA and (m/z)⁻ 469 for the IS. The method was validated in the concentration range of 2.5 − 1470 ng mL⁻¹ for plasma samples and 20 − 11760 ng g⁻¹ for tissue homogenates. The intra- and inter-day assay of precision in plasma and tissues ranged from 1.6% to 7.1% and 3.7% to 9.0%, respectively, and the intra- and inter-day assay accuracy was 84.2 − 106.9% and 82.1 − 108.1%, respectively. Recoveries in plasma and tissues ranged from 83.2% to 106.2%. The limits of detections were 0.5 ng mL⁻¹ or 4.0 ng g⁻¹. The recoveries for all samples were >90%, except for liver, which indicated that ursolic acid may metabolize in liver. The main pharmacokinetic parameters obtained were T _max = 0.42 ± 0.11 h, C _max = 1.10 ± 0.31 μg mL⁻¹, AUC = 1.45 ± 0.21 μg h mL⁻¹ and K _a = 5.64 ± 1.89 h⁻¹. The concentrations of UA in rat lung, spleen, liver, heart, and cerebellum were studied for the first time. This method is validated and could be applicable to the investigation of the pharmacokinetics and tissue distribution of UA in rats.     

Simultaneous Determination of Olanzapine and Fluoxetine by HPLC

A rapid, specific reversed phase HPLC method has been developed for simultaneous determination of olanzapine and fluoxetine in their formulations. Chromatographic separation of these two pharmaceuticals was carried out on an Inertsil C₁₈ reversed phase column (150 mm × 4.6 mm, 5 μm) with a 40:30:30 (v/v/v) mixture of 9.5 mM sodium dihydrogen phosphate (pH adjusted to 6.8 ± 0.1 with triethylamine), acetonitrile and methanol as mobile phase. The flow rate 1.2 mL min⁻¹ and the analytes are monitored at 225 nm. Paroxetine was used as internal standard. The assay results were linear from 25 to 75 μg mL⁻¹ for olanzapine (r ² ≥ 0.995) and 100–300 μg mL⁻¹ for fluoxetine (r ² ≥ 0.995), showed intra- and inter-day precision less than 1.0%, and accuracy of 97.7–99.1% and 97.9–99.0%. LOQ was 0.005 and 0.001 μg mL⁻¹ for olanzapine and fluoxetine, respectively. Separation was complete in less than 10 min. Validation of the method showed it to be robust, precise, accurate and linear over the range of analysis.     

Liquid chromatographic determination of meloxicam in serum after solid phase extraction

A liquid chromatographic method for the determination of meloxicam in serum has been developed. The technique includes a solid phase extraction of the serum samples on [poly (divinylbenzeneco-N-vinylpyrrolidone)] as a solid phase extraction sorbent. After conditioning, the cartridge was loaded with 1 mL of acidified serum containing an internal standard. Elution was carried out using 1 mL of water-acetonitrile (φ _r = 1: 1) mixture. After evaporation of the eluate to dryness and reconstitution of the residue with 0.1 mL of methanol, the samples were analyzed on a Symmetry C18 column. Mobile phase consisted of 1 % aqueous acetic acid/THF/acetonitrile (φ _r = 60: 30: 10) + 0.1 mL of 1-octane sulfonic acid. Detection was carried out using a photodiode array detector. Full validation of the proposed method is provided. Linearity of the method was proven over the range of 0.01–10 φg mL⁻¹ of meloxicam. Meloxicam assay was accurate and reliable with average intra- and inter-day precisions lower than 5.0 % and the intra- and inter-day accuracy higher than 97 %. Limits of detection (LOD) and quantitation (LOQ) found were 0.003 μg mL⁻¹ and 0.01 μg mL⁻¹, respectively. The proposed method was successfully utilized to quantify meloxicam in serum.     

HPLC with Column Switching Coupled to APCI–MS for Pharmacokinetic Study of Amygdalin in Rabbit Plasma

A fast and validated assay was established for the pharmacokinetic study of amygdalin in Armeniacae Semen in rabbit. The method involved column switching (CS) enrichment, separation, post-column derivatization, and atmospheric pressure chemical ionization (APCI) mass spectrometric detection. Plasma sample was enriched by CS using a MAYI-ODS as the first column. Analytes of interest were isolated and analyzed on a second column of Zorbax SB-C₁₈. To detect amygdalin in plasma samples, a T-piece was connected between the HPLC outlet and the APCI source to add a mixture of dichloromethane and methanol to the eluent by an isocratic pump. Calibration graphs showed good linearity over a range of 1.0–1,280 ng mL⁻¹. The detection limit was 0.2 ng mL⁻¹. The intra- and inter-day accuracies were within 3.9%. The method was successfully applied to a study of the pharmacokinetics of amygdalin after an intravenous injection of amygdalin extracts to rabbits with a dose of 400 mg kg⁻¹. The results indicate that amygdalin is a one-compartment open model with a first order absorption phase.     

Simultaneous Determination of Baicalin, Baicalein, Wogonin, Oxysophocarpine, Oxymatrine and Matrine in the Chinese Herbal Preparation of Sanwu-Huangqin-Tang by Ion-Paired HPLC

A sensitive and reliable ion-paired high-performance liquid chromatographic method has been established for the simultaneous quantification of six major active ingredients, namely baicalin, baicalein, wogonin, oxysophocarpine, oxymatrine and matrine in the Chinese herbal preparation, Sanwu-Huangqin-Tang. HPLC analyses were performed on a Phenomenex luna C₁₈ column with mobile phase of methanol–acetonitrile–aqueous phosphoric acid at a flow rate of 0.9 mL min⁻¹. The complete separation was achieved within 35 min for the six target constituents. A good linear regression relationship between peak-areas and concentrations was obtained over the range of 12.10–242.0 μg*mL⁻¹ for baicalin, 5.05–101.0 μg*mL⁻¹ for baicalein, 0.95–19.0 μg*mL⁻¹ for wogonin, 2.75–55.0 μg*mL⁻¹ for oxysophocarpin, 2.75–55.0 μg*mL⁻¹ for oxymatrine and 4.90–98.0 μg*mL⁻¹ for matrine, respectively. The repeatability was evaluated by intra- and inter-day assays with relative standard deviation (RSD) being less than 5.1%. The recoveries, measured at three concentration levels, varied from 93.8 to 102.1%. The assay was successfully applied for determination of six bioactive compounds in Sanwu-Huangqin-Tang. The interaction of chemical constituents was observed when the herbs were used in compatibility. The results indicated that the developed assay method was rapid, accurate and could be readily utilized as a quality control method for Sanwu-Huangqin-Tang.     

Development and Validation of an HPLC Method for Determination of Ceftiofur Sodium

An isocratic high-performance liquid chromatographic method has been developed for assay of ceftiofur sodium in drug substance and in sterile powder for injection. Chromatography was performed on a 250 mm × 4.6 mm, 5 μm particle, C₁₈ column with a 78:22 (v/v) mixture of 0.02 m disodium hydrogen phosphate buffer (pH adjusted to 6.0 with 85% orthophosphoric acid) and acetonitrile as mobile phase, at a flow rate of 1.0 mL min⁻¹. The separation was monitored by UV detection at 292 nm. Validation of the method for linearity and range, intra- and inter-day precision, accuracy, specificity, recovery, robustness, and limits of quantification and detection yielded good results. The calibration plot was linear from 20.0–120.0 μg mL⁻¹ and the correlation coefficient was 0.9999. It was shown that ceftiofur was degraded under acidic, alkaline, oxidative, and photolytic conditions. The method was found to be stability-indicating and could be used for routine analysis of ceftiofur sodium for injection.     

Quantitative Determination of Acyclovir in Aqueous Humor by LC-MS

A specific, sensitive and precise liquid chromatographic assay method was established using LC-MS for the determination of acyclovir (ACV) in aqueous humor (AH), which was directly injected onto an Inertsil ODS-3 C₁₈ column without any pretreatment. The Agilent 1100 series LC–MS system was operated under the electrospray ionization mode (ESI). The analyte was separated from endogenous substances with a mobile phase of methanol: water: acetic acid (5:95:0.1, v/v) at a flow-rate of 0.3mL min⁻¹. A linear response was observed over the concentration range from 5 to 50ng mL⁻¹ (r=0.9993). Intra- and inter-day coefficients of variation were in the ranges 5.2–9.0% and 5.8–8.2%, respectively. The recovery was > 91.0%, and the limit of detection was approximate 1ng mL⁻¹. The pharmacokinetics of topically applied eye-drop and thermosetting gel were compared in rabbits utilizing the present method, the results demonstrated that LC-MS was a powerful tool for the detection of ACV in AH.     

Simultaneous Determination of Triptolide and Tripdiolide in Extract of Tripterygium wilfordii Hook. f. by LC–APCI-MS

A novel method using liquid chromatography coupled with atmospheric pressure chemical ionization mass spectrometry in the negative selected ion monitoring mode has been developed and validated for rapid simultaneous determination of triptolide and tripdiolide in the extract of Tripterygium wilfordii Hook. f. The molecular ions m/z [M–H]⁻ 359 and 375 were selected for the quantification in selected ion monitoring mode for triptolide and tripdiolide. Standard calibration curve was linear over the concentration range of 0.12–24 and 0.15–30 μg mL⁻¹ for triptolide and tripdiolide. The relative standard deviations of intra- and inter-day were in the range of 4.7–9.9 and 8.9–12.6%. The average recoveries were between 96.4 and 104.6%. The limits of quantitation were 2.0 × 10⁻³ and 2.5 × 10⁻³ μg mL⁻¹ for triptolide and tripdiolide.     

High-performance liquid chromatographic assay for simultaneous determination of tramadol and its active metabolite in human plasma. Application to pharmacokinetic studies

Summary A sensitive liquid chromatographic assay for the quantitative determination of the opioid analgesic tramadol and its active metabolite is described. Fluconazole was used as internal standard. The assay involved a singletert-butyl methyl ether extraction and LC analysis with fluorescence detection. Chromatography was at 30°C pumping an isocratic mobile phase of acetonitrile-water (19∶81, v/v) containing 0.06M NaH₂PO₄ and 0.05M triethylamine, adjusted to pH 7.90, at 1 mL min⁻¹ through a reversed-phase, 250×4 mm base-stable column. The limit of quantitation of tramadol and its active metabolite was 1 ng mL⁻¹, only 0.5 mL plasma sample was required for the determination. The calibration curve was linear from 1–1000 ng mL⁻¹. Intra and inter-day precision (C.V.) did not exceed 10%. Mean recoveries of 96.38% for tramadol and 96.62% forO-demethyltramadol with CVs of 0.43% and 1.46% were obtained. Applicability of the method was demonstrated by a pharmacokinetic study on normal volunteers who received 100 mg tramadol intravenously.     

LC Determination of Four Isoflavone Aglycones in Red Clover (Trifolium pratense L.)

Red clover (Trifolium pratense L.) is an important forage plant that contains the isoflavones daidzein, genistein, formononetin, and biochanin A. These compounds have been studied lately due to their human health benefits. The aim of this study was to develop and validate an HPLC method with simplified sample preparation to quantify daidzein, genistein, formononetin and biochanin A simultaneously in red clover leaves. The validation showed that the method is specific, accurate, precise and robust, not to mention that the sample preparation is easier and faster than those described earlier. The response was linear over a range of 0.01–0.2 μg mL⁻¹ for daidzein, 0.05–0.5 μg mL⁻¹ for genistein, 4–40 μg mL⁻¹ for formononetin and 2–20 μg mL⁻¹ for biochanin A. The range of recoveries was 85.6–101.0%. The RSD for intra- and inter-day precision were <2.54 and <7.22%, respectively. Five populations of red clover, from the National Plant Germplasm System-USDA were analyzed and the content of daidzein, genistein, formononetin and biochanin A ranged from 7.87–91.31, 51.60–131.30, 6568.33–23461.82, to 2499.55–10337.33 μg g⁻¹ of dried material, respectively.     

Quantitative Analysis of Triptolide in Human Whole Blood by LC–APCI-IT-MS-MS

In this study, the development and validation of an analytical method for triptolide in whole blood using high-performance liquid chromatography coupled with atmospheric-pressure chemical ionization ion trap tandem mass spectrometry (LC–APCI-IT-MS-MS) is reported. This is the first report of the systematic development and validation of an LC–MS-MS method for the quantitation of triptolide in human whole blood using prednisolone as an internal standard (IS). Prior to LC–MS-MS analysis, liquid–liquid extraction with ethyl acetate was used to isolate them from the biological matrix. Validation parameters such as specificity/selectivity, limit of quantitation (LOQ), linearity, precision, accuracy and stability are evaluated for this method. The calibration curve was linear (r ² = 0.9973) in the concentration range of 0.5–100.0 ng mL⁻¹ in human whole blood with a lower limit of quantitation of 0.5 ng mL⁻¹. Intra- and inter-day relative standard deviations (RSDs) were less than 8.6 and 11.7%, respectively. Extraction recoveries of triptolide ranged from 81.5 to 88.1%. This assay can be used to determine trace triptolide in human whole blood.     

Analysis of 5-hydroxy-N-methyl-2-pyrrolidone and 2-hydroxy-N-methylsuccinimide in plasma

Summary A method for the determination of 5-hydroxy-N-methyl-2-pyrrolidone (5-HNMP) and 2-hydroxy-N-methylsuccinimide (2-HMSI) in plasma was developed. 5-HNMP and 2-HMSI are metabolites to the widely used organic solvent N-methyl-2pyrrolidone (NMP). The 5-HNMP and 2-HMSI were purified from plasma by C8 solid phase extraction, derivatised by bistrimethylsilyl trifluoroacetamid, and analysed by gas chromatography with mass spectrometric detection. For 5-HNMP, the precision was 2–7 % (120 and 780 ng mL⁻¹) and the detection limit was 6 ng mL⁻¹ (m/z 98). For 2-HMSI, the precision was 2–9 % (160 and 1000 ng mL⁻¹) and the detection limit was 4 ng mL⁻¹ (m/z 144). The method is applicable for analysis of plasma samples from workers exposed to NMP.     

Study of a toxin–alkaline phosphatase conjugate for the development of an immunosensor for tetrodotoxin determination

This paper describes a direct competitive immunoenzymatic spectrophotometric assay (ELISA) for tetrodotoxin (TTX) determination and the adaptation of this method for use in an electrochemical assay format. The novelty of this work involves the use of the antigen labelled with alkaline phosphatase (AP); this conjugate was prepared in our laboratory as there is no commercially available conjugate of any kind for TTX. The new conjugate was characterized in terms of its affinity for the specific antibody as well as the residual concentration and the residual activity of the enzyme (AP) incorporated as label. The proposed method based on the new conjugate showed satisfactory results for TTX determination: for the spectrophotometric method the dynamic range was 4–15 ng mL⁻¹ with a limit of detection (LOD) of 2 ng mL⁻¹ (R=0.9247), whereas for the electrochemical protocol the dynamic range was 2–50 ng mL⁻¹ and the LOD was1 ng mL⁻¹.     

Study of the metabolism on tobacco-specific N-nitrosamines in the rabbit by solid-phase extraction and liquid chromatography–tandem mass spectrometry

Metabolism of four tobacco-specific N-nitrosamines (TSNAs), N′-nitrosonornicotine (NNN), N′-nitrosoanatabine (NAT), N′-nitrosoanabasine (NAB), and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) has been studied by solid-phase extraction (SPE) and liquid chromatography–tandem mass spectrometry (LC–MS–MS). 4-(Methylnitrosamino)-4-(3-pyridyl)-1-butanol (iso-NNAL) was used as internal standard. SPE and LC–MS–MS was found to be a rapid, simple, sensitive, and selective method for analysis of TSNAs in rabbit serum. The relative standard deviation (R.S.D., n = 6) for analysis of 5 ng mL⁻¹ and 0.5 ng mL⁻¹ standards and of serum sample spiked with 5 ng mL⁻¹ standards of five TSNAs was 2.1–11% and recovery of 5 ng mL⁻¹ standards from serum was 100.2–112.9%. A good linear relationship was obtained between peak area ratio and concentration in the range of 0.2–100 ng mL⁻¹ for NNAL and 0.5–100 ng mL⁻¹ for other four TSNAs, with correlation coefficients (R ²) >0.99 (both linear and log–log regression). Detection limits for standards in solvent were between 0.04 and 0.10 ng mL⁻¹. Doses of TSNAs administered to rabbits via the auricular vein were 4.67 μg kg⁻¹ and 11.67 μg kg⁻¹, in accordance with the different levels in cigarettes. Metabolic curves were obtained for the four TSNAs and for 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), a metabolite of NNK; on the basis of these curves we modeled metabolic kinetic equations for these TSNAs by nonlinear curve fitting.