Improved method for routine determination of nicotine and its main metabolites in biological fluids

Extrelut^R extraction and glass capillary gas chromatography were applied to the routine determination of nicotine and its metabolites cotinine, nicotine-1′-N-oxide and cotinine-1-N-oxide in urine and plasma. After extraction of nicotine and cotinine both N-oxides and phendimetrazine-N-oxide (used as internal standard) were reduced to their bases by SO₂ on-column and eluted by a mixture of diethyl ether and dichloromethane. The minimum detectable concentrations are 0.03 μg/ml for urinary nicotine and cotinine and 0.1 μg/ml for the N-oxides. In plasma samples the corresponding values are 5 ng/ml and 15 ng/ml, respectively, with sample values as small as 2 ml. The advantage of the direct determination of all four compounds of interest in one sample reduced the amount of plasma required. The straightforward and rapid extraction and reduction procedure as well as the long-term stability of the gas chromatographic separation system make the method suitable for routine application.     

Simultaneous determination of norethisterone and six metabolites in human plasma by capillary gas chromatography with mass-selective detection

A method for the simultaneous determination of norethisterone (NET) and six metabolites in human plasma by capillary gas chromatography-mass-selective detection (GC-MS) is described. The compounds are determined in plasma after enzymatic hydrolysis. After addition of norgestrel as the internal standard, the compounds are extracted from plasma at pH 5 using an Extrelut column and elution with dichloromethane. After evaporation, the compounds are converted into bistrimethylsilyl derivatives which are determined by gas chromatography using a mass-selective detector at m/z 429 for the two dihydro-NET (5β-NET and 5α-NET), m/z 431 for the four tetrahydro-NET (3α,5α-NET, 3α,5β-NET, 3β,5β-NET and 3β,5α-NET), m/z 442 for NET and m/z 456 for the internal standard. The reproducibility and accuracy of the method were found suitable over the range of concentrations between 0.50 and 8 ng/ml for NET, and metabolites except for 5α-dihydro-NET (between 1 and 8 ng/ml). The method was applied to clinical samples.     

Analysis of methadone and metabolites in biological fluids with gas chromatography—mass spectrometry

The analysis of methadone and its metabolites in biological fluids by gas chromatography—mass spectrometry is described with deuterated methadone and metabolites as internal standards. The method allowed the determination of 20 ng methadone in 0.5 ml of plasma or saliva. Mean saliva to plasma ratio of methadone for two patients was determined to be 0.51 ± 0.13. Methadone and 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) in urine were measured by selected ion monitoring. Gas chromatography—mass spectrometry was found to have advantages over conventional gas chromatographic methods in terms of ratio analysis. 1,5-Dimethyl-3,3-diphenyl-2-pyrrolidone previously reported as a metabolite was shown to result primarily from the decomposition of EDDP free base.     

Solid-phase extraction and high-performance liquid chromatographic determination of tamoxifen and its major metabolites in plasma

Tamoxifen (TAM) is a triphenylethylene anti-oestrogen, commonly used in the treatment of breast cancer. Patients receiving tamoxifen therapy may experience both de novo and acquired resistance. As one of the mechanisms for this may be extensive peripheral bio-transformation of tamoxifen, there has been considerable interest in the pharmacokinetics and metabolism of tamoxifen. A reversed-phase high-performance liquid chromatography separation has been developed to determine the levels of tamoxifen and its major metabolites in human plasma. The method is highly sensitive (2 ng/ml) and selective for tamoxifen, cis-tamoxifen (CIS), 4-hydroxytamoxifen (4-OH) and desmethyltamoxifen (DMT). A μBondapak C₁₈ 10 μm column (30 cm × 3.9 mm I.D.) was used, with a mobile phase of methanol-1% triethylamine at pH 8 (89:11, v/v). Sample preparation was carried out using a C₂ (500 mg sorbent, 3 ml reservoirs) solid phase extraction method, and extraction efficiencies were approximately 60% for TAM and its metabolites. Accuracy and precision, as determined by spiking plasma samples with a mixture of tamoxifen and its metabolites, ranged from 85–110% (± 5–10%) at 1 μg/ml, 101–118% (± 8–20%) at 0.1 μg/ml and 111–168% (± 43–63%) at 0.01 μg/ml. Results from 59 patients show mean values of 54 ng/ml for 4-OH; 190 ng/ml for DMT; 93 ng/ml for TAM and 30 ng/ml for CIS (detected in three patients only). This methodology can be applied routinely to the determination of TAM and its metabolites in plasma from patients undergoing therapy.     

Enantioselective determination of mexiletine and its metabolites p-hydroxymexiletine and hydroxymethylmexiletine in rat plasma by normal-phase liquid chromatography-tandem mass spectrometry: application to pharmacokinetics

Mexiletine (MEX), hydroxymethylmexiletine (HMM) and p-hydroxymexiletine (PHM) were analyzed in rat plasma by LC-MS/MS. The plasma samples were prepared by liquid-liquid extraction using methyl-tert-butyl ether as extracting solvent. MEX, HMM, and PHM enantiomers were resolved on a Chiralpak(R) AD column. Validation of the method showed a relative standard deviation (precision) and relative errors (accuracy) of less than 15% for all analytes studied. Quantification limits were 0.5 ng ml(-1) for the MEX and 0.2 ng ml(-1) for the HMM and PHM enantiomers. The validated method was successfully applied to quantify the enantiomers of MEX and its metabolites in plasma samples of rats (n = 6) treated with a single oral dose of racemic MEX.     

Determination of the anti-platelet-activating factor BN-50727 and its metabolites in human plasma by high-performance liquid chromatography-solid-phase extraction

A sensitive and selective HPLC-solid-phase extraction procedure was developed for the determination of platelet-activating factor antagonist BN-50727 and its metabolites in human plasma. The procedure consisted of an automated solid-phase extraction of the drug and metabolites on disposable propylcarboxylic acid cartridges, followed by on-line chromatographic separation. The method was linear from 3.75 to 2400 ng/ml and the limit of quantitation for BN-50727 in plasma samples was 3.75 ng/ml. The within-run precision of the method, expressed as relative standard deviation, ranged from 2.1 to 8.1%. The accuracy, expressed as relative error, ranged from −3.5 to 4.0%. For the main metabolite, the O-demetthylated BN-50727 product, the method was linear from 7.5 to 2400 ng/ml and the limit of quantitation in plasma was 7.5 ng/ml. The within-run precision ranged from 2.1 to 11.0% and the accuracy from −5.3 to 1.1%. This paper describes the validation of the analytical methodology for the determination of BN-50727 in human plasma and also of its metabolites. The method has been used to follow the time course of BN-50727 and its metabolites in human plasma after administration of single and multiple doses.     

Simple and sensitive high-performance liquid chromatographic method for the determination of diltiazem and six of its metabolites in human plasma

A sensitive, specific and reproducible high-performance liquid chromatographic technique is described for the simultaneous determination in human plasma of diltiazem (DZ) and six of its primary and secondary metabolites which are products of N- and O-demethylation, deacetylation and N-oxidation. The method involves addition of excess KHCO₃ to 1 ml of plasma, followed by extraction with 4 ml of ethyl acetate. The organic layer was extracted with 0.01 M HCl and the aqueous layer was dried under nitrogen and then reconstituted with 0.002 M HCl. DZ and its metabolites were free from interference and were baseline-separated. Calibration curves were linear in the concentration range studied (5–500 ng/ml for all the species). The lower limit of quantification of the assay was 5 ng/ml for DZ and the metabolites. Inter-day and intra-day coefficients of variation were less than 10%. The applicability of this procedure is shown by evaluating the kinetics of DZ and its metabolites in three patients receiving chronic DZ therapy. N-Demethyldiltiazem, deacetyldiltiazem and N-demethyldeacetyldiltiazem were found to be the major metabolites, as previously described. Deacetyldiltiazem N-oxide was found in two of the patients. The other two known but unreported metabolites in human, O-demethyldeacetyldiltiazem and N,O-didemethyldeacetyldiltiazem, were found in the plasma of all three patients.     

Determination of dioxopiperazine metabolites of quinapril in biological fluids by gas chromatography—mass spectrometry

The dioxopiperazine metabolites of quinapril in plasma and urine were extracted with hexane—dichloroethane (1:1) under acidic conditions. Following derivatization with pentafluorobenzyl bromide and purification of the desired reaction products using a column packed with silica gel, the metabolites were analysed separately by capillary column gas chromatography—electron-impact mass spectrometry with selected-ion monitoring. The limits of quantitation for the metabolites were 0.2 ng/ml in plasma and 1 ng/ml in urine. The limits of detection were 0.1 ng/ml in plasma and 0.5 ng/ml in urine, at a signal-to-noise ratio of > 3 and > 5, respectively. The proposed method is applicable to pharmacokinetic studies.     

Simultaneous determination of isosorbide dinitrate and its mononitrate metabolites in human plasma by capillary gas chromatography with electron-capture detection

A method for the simultaneous determination of isosorbide dinitrate (ISDN) and its mononitrate metabolites (2- and 5-ISMN) in human plasma by capillary gas chromatography with electron-capture detection was developed. Two internal standards were used: isomannide dinitrate (IMDN) for the determination of ISDN and isomannide mononitrate (IMMN) for the determinations of 2- and 5-ISMN. After addition of the internal standards, the compounds were isolated from plasma by solid-liquid extraction. They were determined by gas chromatography using an electron-capture detector. The reproducibility and accuracy of the method were found suitable in the range of concentrations 2.5–83 ng/ml for ISDN, 2.6–208 ng/ml for 2-ISMN and 2.3–1010 ng/ml for 5-ISMN. The limit of quantitation (LOQ) was about 2.5 ng/ml for each compound. The method was applied to clinical samples.     

Mass fragmentographic determination of lofepramine and its metabolites in human plasma and urine using deuterated internal standards

A sensitive and specific method for the determination of lofepramine and its metabolites, desipramine and 2-hydroxydesipramine, in human plasma and urine is described. Lofepramine, desipramine and 2-hydroxydesipramine were derivatized to ethyl p-chlorobenzoate, the bis(heptafluorobutyryl) derivative and the N,O-bis(trifluoroacetyl) derivative, respectively, and then analysed by gas chromatography—mass fragmentography. Corresponding deuterated compounds were used as internal standards. Determination was possible at levels as low as 2 ng/ml for lofepramine and desipramine and 20 ng/ml for 2-hydroxydesipramine.     

Simultaneous determination of a novel calcium entry blocker, monatepil maleate, and its metabolites in rat plasma by means of solid-phase extraction and reversed-phase liquid chromatography with electrochemical detection

A reversed-phase LC method with electrochemical detection is described for the simultaneous determination of monatepil maleate (AJ-2615, AJ), a novel calcium entry blocker, and its three S-oxidiized metabolites in plasma. These compounds were extracted from plasma by solid-phase extraction and injected onto an ODS column. The determination limit in plasma (0.5 ml) was 10 ng/ml for AJ and 5 ng/ml for the three metabolites. The metthod was applied to the determination of AJ and the metabolites in rat plasma samples.     

Determination of clozapine and its major metabolites in human serum using automated solid-phase extraction and subsequent isocratic high-performance liquid chromatography with ultraviolet detection

An isocratic high-performance liquid chromatographic (HPLC) method with ultraviolet detection is described for the quantification of the atypical neuroleptic clozapine and its major metabolites, N-desmethylclozapine and clozapine N-oxide, in human serum or plasma. The method included automated solid-phase extraction on C₁₈ reversed-phase material. Clozapine and its metabolites were separated by HPLC on a C₁₈ ODS Hypersil analytical column (5 μm particle size; 250 mm × 4.6 mm I.D.) using an acetonitrile—water (40:60, v/v) eluent buffered with 0.4% (v/v) N,N,N′,N′-tetramethylethylenediamine and acetic acid to pH 6.5. Imipramine served as internal standard. After extraction of 1 ml of serum or plasma, as little as 5 ng/ml of clozapine and 10 or 20 ng/ml of the metabolites were detectable. Linearity was found for drug concentrations between 5 and 2000 ng/ml as indicated by correlation coefficients of 0.998 to 0.985. The intra- and inter-assay coefficients of variation ranged between 1 and 20%. Interferences with other psychotropic drugs such as benzodiazepines, antidepressants or neuroleptics were negligible. In all samples, collected from schizophrenic patients who had been treated with daily oral doses of 75–400 mg of clozapine, the drug and its major metabolite, N-desmethylclozapine, could be detected, while the concentrations of clozapine N-oxide were below 20 ng/ml in three of sixteen patients. Using the method described here, data regarding relations between therapeutic or toxic effects and drug blood levels or metabolism may be collected in clinical practice to improve the therapeutic efficacy of clozapine drug treatment.     

Gas—liquid chromatographic determination of flurazepam and its major metabolites in plasma with electron-capture detection

A sensitive and selective gas—liquid chromatographic method, using the electron-capture detector for the quantitative determination of flurazepam and its major blood metabolites is described. After extraction and back-extraction steps, flurazepam (I) is well separated from its main metabolites, N-1-hydroxyethylflurazepam (metabolite II) and N-1-desalkylflurazepam (metabolite III). Metabolite II is quantitated after forming its stable tert-butyldimethylsilyl derivative by reaction with tert-butyldimethylchlorosilane—imidazole reagent. The procedure permits the rapid and selective routine determination of flurazepam and its metabolites (II and III) in plasma with a detection limit of 3 ng/ml for flurazepam (I), 1 ng/ml for metabolite II and 0.6 ng/ml for metabolite III. The procedure is linear over the range of concentrations encountered after administration of a single oral therapeutic dose. No interference from the biological matrix is apparent. The suitability of the method for the analysis of biological samples was tested by studying the variation with time of flurazepam and its metabolites' plasma concentrations in normal human volunteers after a single, therapeutic 30-mg oral dose of flurazepam.     

Simultaneous determination of nefiracetam and its metabolites by high-performance liquid chromatography

A reversed-phase high-performance liquid chromatographic assay was developed to simultaneously quantitate nefiracetam (NEF), a novel nootropic agent, and its three known oxidized metabolites (N-[(2,6-dimethylphenylcarbamoyl)methyl]succinamic acid (5-COOH-NEF), 4-hydroxy-NEF and 5-hydroxy-NEF) in human serum and urine. The quantitative procedure was based on solid-phase extraction with Sep-Pak C₁₈ and ultraviolet detection at 210 nm. The calibration curves of NEF and the metabolites were linear over a wide range of concentrations (0.5–21.5 nmol/ml for NEF and 0.4–9.5 nmol/ml for metabolites in serum and 4–86 nmol/ml for NEF and 8–190 nmol/ml for metabolites in urine). Intra- and inter-day assay coefficients of variation for the compounds were less than 10%. The limit of detection was 0.1 nmol/ml for NEF, 5-COOH-NEF and 4-hydroxy-NEF, and 0.2 nmol/ml for 5-hydroxy-NEF in both serum and urine. This method is applicable for the determination of NEF and its metabolites in human serum and urine with satisfactory accuracy and precision.     

Simultaneous quantitative determination of cilostazol and its metabolites in human plasma by high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) method for the simultaneous determination of cilostazol, a quinolinone derivative, and its known metabolites OPC-13015, OPC-13213, OPC-13217, OPC-13366, OPC-13269, OPC-13326 and OPC-13388 in human plasma was developed and validated. Cilostazol, its metabolites and two internal standards, OPC-3930 and OPC-13112, were extracted from human plasma by a combination of liquid–liquid and liquid–solid phase extractions, with combined organic solvents of n-butanol, methanol, chloroform, methyl-tert.-butyl ether, and a Sep-Pak silica column. The combined extract was then evaporated and the residue was reconstituted in ammonium acetate buffer (pH 6.5). The reconstituted solution was injected onto a HPLC system and was subjected to reversed-phase HPLC on a 5 μm ODS-80TM column to obtain quality chromatograph and good peak resolution. A gradient mobile phase with different percentages of acetonitrile in acetate buffer (pH 6.5) was used for the resolution of analytes. Cilostazol, its metabolites and the two internal standards were well separated at baseline from each other with resolution factor being 74 and 138. This HPLC method was demonstrated to be specific for all analytes of interest with no significant interference from the endogenous substances of human plasma. The lower limit of quantitation was 20 ng/ml for cilostazol and all metabolites. The method was validated initially for an extended linear range of 20–600 ng/ml for all metabolites and cilostazol, and has been revised later for a linear range of 20–1200 ng/ml for cilostazol and two major and active metabolites OPC-13015 and OPC-13213. The overall accuracy (relative recovery) of this method was established to be 98.5% to 104.9% for analytes with overall precision (CV) being 1.5% to 9.0%. The long-term stability of clinical plasma samples was established for at least one year at −20°C. Two internal standards of OPC-3930 and OPC-13112 were evaluated and validated. However, the data indicated that there was no significant difference for all accuracy and precision obtained by using either OPC-3930 or OPC-13112. OPC-3930 was chosen as the internal standard for the analysis of plasma samples from clinical studies due to its shorter retention time. During the validation standard curves had correlation coefficients greater than or equal to 0.998 for cilostazol and the seven metabolites. These data clearly demonstrate the reliability and reproducibility of the method.     

Determination of clozapine, desmethylclozapine and clozapine N-oxide in human plasma by reversed-phase high-performance liquid chromatography with ultraviolet detection

An isocratic high-performance liquid chromatography (HPLC) method with ultraviolet detection for the simultaneous determination of clozapine and its two major metabolites in human plasma is described. Analytes are concentrated from alkaline plasma by liquid–liquid extraction with n-hexane–isoamyl alcohol (75:25, v/v). The organic phase is back-extracted with 150 μl of 0.1 M dibasic phosphate (pH 2.2 with 25% H₃PO₄). Triprolidine is used as internal standard. For the chromatographic separation the mobile phase consisted of acetonitrile–0.06 M phosphate buffer, pH 2.7 with 25% phosphoric acid (48:52, v/v). Analytes are eluted at a flow-rate of 1.0 ml/min, separated on a 250×4.60 mm I.D. analytical column packed with 5 μm C₆ silica particles, and measured by UV absorbance detection at 254 nm. The separation requires 7 min. Calibration curves for the three analytes are linear within the clinical concentration range. Mean recoveries were 92.7% for clozapine, 82.0% for desmethylclozapine and 70.4% for clozapine N-oxide. C.V. values for intra- and inter-day variabilities were ≤13.8% at concentrations between 50 and 1000 ng/ml. Accuracy, expressed as percentage error, ranged from −19.8 to 2.8%. The method was specific and sensitive with quantitation limits of 2 ng/ml for both clozapine and desmethylclozapine and 5 ng/ml for clozapine N-oxide. Among various psychotropic drugs and their metabolites, only 2-hydroxydesipramine caused significant interference. The method is applicable to pharmacokinetic studies and therapeutic drug monitoring.     

Determination of angiotensin metabolites in human plasma by fluorimetric high-performance liquid chromatography using a heart-cut column-switching technique

Fluorimetric column-switching HPLC method with naphthalene-2,3-dialdehyde (NDA) was developed for the determination of endogenous angiotensin (ANG) metabolites in human plasma. After one-step extraction to clean up the ultrafiltered plasma sample on the reversed HPLC system, the zone of the retention time of each ANG analyte was subjected to the NDA-derivatization. After putting into a first Phe-ODS (for ANG (3–4) and (5–8) determinations) or ODS column (for ANG I and II determinations), the heart-cut of the retention time of the NDA-ANG was separated on a second ODS column with a mobile phase containing 5 mM ion-pair reagent. Complete separation and good detection were accomplished within 2 h. Good linearity of the regression equation for all ANG analytes with the correlation coefficient of >0.993 as well as good reproducibility (C.V.<4.0%). Good agreement of the range of ANG II plasma level between the present (25–47 fmol/ml in plasma) and the radioimmunoassay methods (28–52 fmol/ml in plasma) indicated that the column-switching method could be applicable for the determination of endogenous smaller ANGs as well as for ANG I or II in plasma.     

Determination of plasma concentrations of epirubicin and its metabolites by high-performance liquid chromatography during a 96-h infusion in cancer chemotherapy

In order to determine epirubicin and its metabolites at low concentrations (<38 ng/ml) in small plasma samples, a fast reliable method based on a precipitation pre-treatment and sensitive reversed-phase isocratic HPLC has been developed and validated for epirubicin in the range 5–100 ng/ml. The R.S.D. was 5–9% over this concentration range. For human serum containing 25 ng/ml of epirubicin, the inter- and intra-day variation was <10%. Recoveries of the metabolites epirubicinol, 7-deoxydoxorubicinone and 7-deoxydoxorubicinolone at 20 ng/ml ranged from 94–104%. The assay has been used to study human plasma samples taken during a 96-h infusion of epirubicin in a patient with multiple myeloma. The combined levels of the unseparated metabolites, epirubicin glucuronide and epirubicinol glucuronide, were semiquantitatively determined after treatment with β-glucuronidase. The metabolites epirubicinol and 7-deoxydoxorubicinolone, but not 7-deoxydoxorubicinone, were also detected and measured.     

Quantitative determination of tulobuterol and its metabolites in human urine by mass fragmentography

A method is described for the simple and simultaneous determination of tulobuterol and its metabolites in human urine by gas chromatography-mass spectrometry. Quantification was achieved by single-ion monitoring at m/e 86 derived from trimethylsilyl-tulobuterol and its metabolites using a column packed with a mixed phase, 2% OV-1–2% QF-1 (1 : 1, w/w). The detection limits were estimated to be 2 ng/ml in urine for tulobuterol and 5 ng/ml for metabolites, respectively.     

Determination of sulfinpyrazone and two of its metabolites in human plasma and urine by gas chromatography and selective detection

A selective and sensitive gas chromatographic method for simultaneous determination of sulfinpyrazone and two of its metabolites (the para-hydroxylated metabolite and the sulfone metabolite) in biological fluids using alkali flame ionization detection (AFID), electron capture detection (ECD) and mass fragmentographic detection is described. The compounds are extracted from the samples, methylated and separated on 2% OV-17 or 8% OV-225 columns. Phenylbutazone is used as internal standard. Standard curves are linear. The coefficient of variation at 10 μg/ml of sulfinpyrazone in plasma was shown to be 1.8% (AFID), and the detection limits were 0.1 μg/ml (AIFD) and 10 ng/ml (ECD). Mass spectra of the methylated compounds are shown and serum concentration curves after oral administration of 100 mg sulfinpyrazone to two persons are determined together with the excreted amounts of drug and metabolites.