Validation and application of a method for the determination of nicotine and five major metabolites in smokers' urine by solid-phase extraction and liquid chromatography-tandem mass spectrometry

An SPE-LC-MS/MS method was developed, validated and applied to the determination of nicotine and five major metabolites in human urine: cotinine, trans-3'-hydroxycotinine, nicotine-N-glucuronide, cotinine-N-glucuronide and trans-3'-hydroxycotinine-O-glucuronide. A 500 microL urine sample was pH-adjusted with phosphate buffer (1.5 mL) containing nicotine-methyl-d3, cotinine-methyl-d3 and trans-3'-hydroxycotinine-methyl-d3 internal standards. For the unconjugated metabolites, an aliquot (800 microL) of the buffered solution was applied to a 30 mg Oasis HLB-SPE column, rinsed with 2% NH4OH/H2O (3.0 mL) and H2O (3.0 mL) and eluted with methanol (500 microL). The eluate was analyzed isocratically (100% methanol) by LC-MS/MS on a diol column (50 x 2.1 mm). For the total metabolites, a beta-glucuronidase/buffer preparation (100 microL) was added to the remaining buffered solution and incubated at 37 degrees C (20 h). An aliquot (800 microL) of the enzymatically treated buffered solution was extracted and analyzed in the same manner. The conjugated metabolites were determined indirectly by subtraction. The quantitation range of the method (ng/mL) was 14-10,320 for nicotine, 15-9800 for cotinine and 32-19,220 for trans-3'-hydroxycotinine. The validated method was used to observe diurnal variations from a smoker's spot urine samples, elimination half-lives from a smoker's 24 h urine samples and metabolite distribution profiles in the spot and 24 h urine samples.     

Automated trace enrichment for screening and/or determination of primary, secondary and tertiary amphetamines in biological samples by liquid chromatography

A rapid and simple liquid chromatographic method for the automated determination of amphetamines in biological fluids was developed. The proposed procedure is based on the injection of 250 microL of sample into a 20 x 2.1 mm id precolumn (packed with a 30 microns Hypersil C18 stationary phase) for enrichment and purification of the analytes. Next, the analytes are transferred to a 5 microns LiChrospher 100 RP18, 125 x 4 mm id analytical column for their separation under reversed-phase conditions. Water was used to eliminate the matrix components from the precolumn and a 0.2 M phosphate buffer (pH 3) containing 2% triethylamine was the mobile phase for the resolution of the amphetamines. The UV detector was set at 210 nm. The method was applied to the determination of different primary, secondary and tertiary amphetamines in plasma and urine: beta-phenylethylamine, norephedrine, ephedrine, N-methylpseudoephedrine, pseudoephedrine, N-methylephedrine, amphetamine, 3-phenylpropylamine and methamphetamine. The method provides satisfactory linearity and reproducibility within the tested concentration range (1.0-10.0 micrograms mL-1) and limits of detection of 50-500 ng/mL-1.     

Direct and sensitive analysis of methamphetamine, ketamine, morphine and codeine in human urine by cation-selective exhaustive injection and sweeping micellar electrokinetic chromatography

Cation-selective exhaustive injection and sweeping micellar electrokinetic chromatography (CSEI-Sweep-MEKC) was directly used to test some abuse drugs in human urine, including morphine (M), codeine (C), ketamine (K) and methamphetamine (MA). First, phosphate buffer (50 mM, pH 2.5) containing 30% methanol was filled into uncoated fused silica capillary (40 cm, 50 microm I.D.), then high conductivity buffer (100 mM phosphate, 6.9 kPa for 99.9 s) was followed. Electrokinetic injection (10 kV, 500 s) was used to load samples and to enhance sensitivity. The stacking step and separation were performed at -20 kV and 200 nm using phosphate buffer (25 mM, pH 2.5) containing 20% methanol and 100 mM sodium dodecyl sulfate. Using CSEI-Sweep-MEKC, the analytes could be simultaneously analyzed and have a detection limit down to ppb level. It was unnecessary to have sample pretreatments. During method validation, calibration plots were linear (r>or=0.9982) over a range of 150-3,000 ng/mL for M and C, 250-5,000 n g/mL for MA, and 50-1,000 ng/mL for K. The limits of detection were 15 ng/mL for M and C, and 5 ng/mL for MA and K (S/N=3, sampling 500 s at 10 kV). Comparing with capillary zone electrophoresis, the results indicated that this stacking method could increase 6,000-fold sensitivity for analysis of MA. Our method was applied for analysis of 28 real urine samples. The results showed good coincidence with immunoassay and GC-MS. This method was feasible for application to detect trace levels of abused drugs in forensic analysis.     

Determination of bisphenol A and 10 alkylphenols in serum using SDS micelle capillary electrophoresis with gamma-cyclodextrin.

A new micelle capillary electrophoresis based on cyclodextrin micellar electrokinetic chromatography (MEKC) for the determination of bisphenol A and 10 alkylphenols in rat serum is reported. Several surfactants and dextrins were studied. Bisphenol A and alkylphenols were separated using a 50 microm x 50 cm capillary with 20 mM borate phosphate buffer (pH 8.0) containing 20 mM sodium dodecylsulfate and 5 mM gamma-cyclodextrin as carrier. The method could determine 0.6-2000 microg/mL of phenols in 100 microL serum by photometric detection at 214 nm. Using 2.0 mL serum, 1.0 ng/mL of phenols could be determined. The relative standards deviations were 6.3-7.7% at 10 microg/mL in serum. The recoveries were 91.8-93.0% with 10 microg/mL serum samples.     

Cation-selective exhaustive injection and sweeping micellar electrokinetic chromatography for analysis of morphine and its four metabolites in human urine

A cation-selective exhaustive injection and sweeping micellar EKC (CSEI-Sweep-MEKC) was established to analyze morphine and its four metabolites, including codeine, normorphine (NM), morphine-3-glucuronide (M3G), and morphine-6-glucuronide (M6G). After SPE, the urine samples were analyzed by this CE method. The phosphate buffer (75 mM, pH 2.5) containing 30% methanol was first filled into an uncoated fused-silica capillary (40 cm, 50 microm id), then a high-conductivity buffer (120 mM phosphate, 10.3 kPa for 99.9 s) followed. The pretreated urine sample was loaded by electrokinetic injection (10 kV, 600 s). The stacking and separation were performed by using phosphate buffer (25 mM, pH 2.5) containing 22% methanol and 100 mM SDS at -20 kV, and detected at 200 nm. During method validation, calibration plots were linear (r > or = 0.998) over a range of 30-3000 ng/mL for morphine, NM, and codeine, 100-2000 ng/mL for M6G, and 80-3200 ng/mL for M3G. The LODs (S/N = 5, sampling 600 s at 10 kV) were 10 ng/mL for morphine, NM, and codeine, 35 ng/mL for M6G, and 25 ng/mL for M3G. This stacking CE method could increase 2500-fold sensitivity of codeine, when comparing with CZE. Five addicts' urine specimens were analyzed. Their results were compared with those of LC-MS-MS, and showed good coincidence. This method could be feasible for monitoring morphine and its metabolites in forensic interest and pharmacokinetic investigations.     

High-performance liquid chromatographic determination of BRB-I-28, a novel antiarrhythmic agent, in dog plasma and urine.

A sensitive reversed-phase high-performance liquid chromatographic (HPLC) technique with ultraviolet detection has been developed to determine the concentration of BRB-I-28 (I), a novel antiarrhythmic agent, in dog plasma and urine. The mobile phase was acetonitrile-methanol-37.5 mM phosphate buffer, pH 6.8-triethylamine (50:50:75:0.1, v/v). The compound was extracted from dog plasma and urine with chloroform after alkalinization with sodium hydroxide. The extraction recovery was 83% from plasma and 84% from urine. Good linearity (r > 0.996) was observed throughout the ranges 0.1-12.0 micrograms/ml (plasma) and 0.1-8.0 micrograms/ml (urine). Intra- and inter-assay variabilities were less than 4%. The lower limit of quantitation was 0.08 microgram/ml in either plasma or urine. HPLC analysis of plasma and urine samples from a dog treated with I has demonstrated that the method was accurate and reproducible.     

Cloud point preconcentration prior to capillary zone electrophoresis: simultaneous determination of platinum and palladium at trace levels

The incorporation of a cloud point extraction (CPE) step prior to capillary electrophoresis (CE) for simultaneously determining platinum and palladium at sub-microg/L levels is presented and evaluated. The analytes were extracted as 2-(5-bromo-2-pyridylazo)-5-diethylaminophenol complexes, at pH 2.0, mediated by micelles of the nonionic surfactant polyethyleneglycolmono-p-nonylphenyl ether (PONPE 7.5). The separation-determination step was developed from 150 microL of the extracted surfactant-rich phase diluted with 50 microL of acetonitrile (ACN). An exhaustive study of the variables affecting the cloud point extraction with PONPE 7.5 and the CZE step was done. The type and composition of the background electrolytes (BGEs) were investigated with respect to separation selectivity, reproducibility, and stability. A BGE of 50 mM monobasic sodium phosphate containing 30% ACN, pH 4.53 was found to be optimal for the separation of metal chelates. Detection was performed at 576 nm. An enrichment factor of 250 was obtained for the preconcentration of 50 mL of sample solution. The detection limits for the preconcentration of 50 mL of sample were 0.04 microg/L for Pt and 0.08 microg/L for Pd. As an analytical demonstration, ultratrace concentrations of platinum and palladium were conveniently quantitated in spiked water and urine samples.     

高效液相色谱法测定吲哚美辛控释胶囊的血药浓度和生物利用度

采用ＯＤＳ柱,甲醇－稀磷酸溶液（７６２４）为流动相,２６０ｎｍ为检测波长,建立了测定血浆中吲哚美辛浓度的高效液相色谱法,并测定了吲哚美辛控释胶囊炎痛康的血药浓度。结果表明,血浆中吲哚美辛浓度在０．１２５～５．０ｍｇ／Ｌ范围内线性关系良好（ｒ＝０．９９９６）,检测限６２．５μｇ／Ｌ（Ｓ／Ｎ＝３１）,平均回收率为１００．４％,日内和日间ＲＳＤ均小于５％。１１位受试者单剂量口服炎痛康后的相对生物利用度为１０２．３８％。     

Quantitative analysis of D-24851, a novel anticancer agent, in human plasma and urine by liquid chromatography coupled with tandem mass spectrometry

The development of a liquid chromatography/tandem mass spectrometric assay for the quantitative analysis of the novel tubulin inhibitor D-24851 in human plasma and urine is described. D-24851 and the deuterated internal standard were extracted from 250 microL of plasma or urine using hexane/ether (1:1, v/v). Subsequently, 10-microL aliquots of reconstituted extracts were injected onto an Inertsil ODS analytical column (50 x 2.0 mm i.d., 5 microm particle size). An eluent consisting of methanol/5 mM ammonium acetate, 0.004% formic acid in water (80:20, v/v) was pumped at a flow rate of 0.2 mL/min. An API 365 triple quadrupole mass spectrometer was used in the multiple reaction monitoring mode for sensitive detection. For human plasma a dynamic range of 1-1000 ng/mL was validated, and for human urine a range of 0.25-50 ng/mL. Validation was performed according to the most recent FDA guidelines and all results were within requirements. The assay has been successfully applied to support a phase I clinical trial with orally administered D-24851.     

Enhanced separation of purine and pyrimidine bases using carboxylic multiwalled carbon nanotubes as additive in capillary zone electrophoresis

This paper describes the enhanced separation of adenine (A), hypoxanthine (HX), 8-azaadenine (8-AA), thymine (T), cytosine (C), uracil (U) and guanine (G) by CZE dispersing carboxylic multiwalled carbon nanotubes (c-MWNTs) into the running buffer. The effect of important factors such as c-MWNT nanoparticle concentration, the acidity and concentration of running buffer, and separation voltage were investigated to acquire the optimum conditions. The seven purine and pyrimidine bases could be well separated within 16 min in a 35 cm effective length fused-silica capillary at a separation voltage of +8.0 kV in a 23 mM tetraborate buffer (pH 9.2) containing 8.0 x 10(-5) g/mL c-MWNTs. Under the optimal conditions, the linear ranges were of 2-250 microg/mL for A (R2 = 0.995), 3-200 microg/mL for U (R2 = 0.990) and G (R2 = 0.992), 3-250 microg/mL for T (R2 = 0.998), 2-200 microg/mL for C (R2 = 0.985) and 4-200 microg/mL for HX (R2 = 0.988) and 8-AA (R2 = 0.990). The detection limits were 0.9 microg/mL for A (S/N = 3), 2.4 microg/mL for U, 2.0 microg/mL for T, 1.5 microg/mL for C, 2.5 microg/mL for G and 3.0 microg/mL for HX and 8-AA. The proposed method was successfully applied for determining five purine and pyrimidine bases in yeast RNA.     

High-performance liquid chromatographic analysis of a novel carbapenem antibiotic in human plasma and urine

High-performance liquid chromatographic methods for quantification of a novel carbapenem anti-infective agent, I, in plasma and urine have been developed, validated, and applied to clinical samples. The carbapenem is stabilized in the matrix by the addition of a non-nucleophilic buffer, rapid freezing, and storage at -70 degrees C. After addition of another carbapenem, II, as internal standard, plasma proteins are precipitated with acetonitrile, which is subsequently extracted from the sample with methylene chloride. A portion of the aqueous phase is injected onto a reversed-phase phenyl column that is eluted with 4% (v/v) acetonitrile in 15 mM ammonium phosphate (pH 7.4). The urine assay entails addition of the internal standard II to buffered urine, which is subsequently extracted with methylene chloride prior to injection of the aqueous phase onto a cation-exchange column. The urine assay mobile phase is 5% v/v tetrahydrofuran in 100 mM sodium acetate (pH 5.4). The detector response at 313 nm is a linear (r greater than 0.99) function of concentration over the ranges 0.50-100 micrograms/ml and 2.0-200 micrograms/ml for the plasma and urine assays, respectively. Thermal degradation products do not interfere with either assay. These assays have proven to be accurate, precise, reproducible, and rugged during clinical sample analyses.     

气相色谱-质谱法测定氯氮平及其去甲基代谢物

建立了测定人血清中氯氮平及其去甲基代谢物的柱前衍生化气相色谱－质谱选择离子监测的分析方法。以三氟乙酸酐作酰化剂,对衍生化条件和样品预处理方法实施了优化。氯氮平和去甲氯氮平的线性范围为１～１２８μｇ／Ｌ,最低检测浓度：氯氮平为０．１μｇ／Ｌ,去甲氯氮平为０．２μｇ／Ｌ,两者的回收率均大于８３％,相对标准偏差都小于１０％。将所建立的方法应用于服用细胞色素氧化酶Ｐ４５０１Ａ２抑制剂前后低剂量氯氮平的药代动力学自身对照试验中,结果显示氯氮平的代谢水平明显受Ｐ４５０１Ａ２活性的影响。     

Capillary electrophoretic chiral determination of mirtazapine and its main metabolites in human urine after enzymatic hydrolysis

Capillary electrophoresis and liquid-phase microextraction using porous polypropylene hollow fibers were employed for the enantioselective analyses of mirtazapine and its metabolites demethylmirtazapine and 8-hydroxymirtazapine in human urine. Before the extraction, urine samples (1.0 mL) were submitted to enzymatic hydrolysis at 37 degrees C for 16 h. Then, the enzyme was precipitated with trichloroacetic acid, the pH was adjusted to 8 with 0.5 mol/L phosphate buffer solution (pH 11) and 15% sodium chloride was further added. The analytes were transferred from the aqueous donor phase, through n-hexyl ether (organic solvent immobilized in the fiber), into 0.01 moL/L acetic acid solution (acceptor phase). The electrophoretic analyses were carried out in 50 mmol/L phosphate buffer solution (pH 2.5) containing 0.55% w/v carboxymethyl-beta-cyclodextrin. The method was linear over the concentration range of 62.5-2500 ng/mL for each mirtazapine and 8-hydroxymirtazapine enantiomer and 62.5-1250 ng/mL for each demethylmirtazapine enantiomer. The quantification limit was 62.5 ng/mL for all the enantiomers. Within-day and between-day assay precision and accuracy were lower than 15% for all the enantiomers. Finally, the method proved to be suitable for pharmacokinetic studies.     

Liquid-phase microextration combined with liquid chromatography-electrospray tandem mass spectrometry for detecting diuretics in urine

Trace amounts of diuretics were determined in human urine by hollow fiber liquid-phase microextraction (LPME) combined with liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) in this study. Chromatography was performed on a C(8) reversed-phase column. A 25 microL n-octanol was used to extract analytes in urine. Extraction was optimized using a pH 2 solution spiked with 0.15 g/mL NaCl for 40 min at 40 degrees C with 1010 rpm stirring. The limits of detection of diuretics in urine were 0.3-6.8 ng/mL, and linearity range was 1-1000 ng/mL. Recoveries of spiked 50 ng/mL diuretics were 97.7-102.5%. The intra-day precision and inter-day precision were 3-18% and 4-21%, respectively. The diuretics concentration profiles in patient urine were also determined. The results of this study reveal the adequacy of LPME-LC-MS/MS method for analyzing diuretics in urine and quantification limits exceed World Anti-Doping Agency requirements.     

Simplified high performance liquid chromatographic quantitation of antipyrine

Antipyrine is used as an index of hepatic drug metabolism. The authors devise a simple and rapid high performance liquid chromatography method to quantitate antipyrine in serum with small volume (50 microliters). Minimal sample preparation is needed and the assay is sensitive to concentrations of 0.2 micrograms/ml. Mobile phase consists of 35% methanol in 67 mM phosphate buffer pH 3.5 with antipyrine detected by monitoring the eluate at 225 and 254 nm. Within-run and run-to-run coefficients of variation are less than or equal to 2.8 and 4.6%, respectively. Recoveries from serum average 100.8%. It is concluded that this technique for antipyrine is simple, rapid, and well characterized.     

Fast HPLC method using ion-pair and hydrophilic interaction liquid chromatography for determination of phenylephrine in pharmaceutical formulations

A rapid procedure based on a direct extraction and HPLC determination with fluorescence detection of phenylephrine in pharmaceutical sachets that include a large excess of paracetamol (65 + 1, w/w), ascorbic acid (5 + 1, w/w), and other excipients (aspartame and sucrose) was developed and validated. The final optimized chromatographic method for ion-pair chromatography used an XTerra RP18 column, 3 microm particle size, 50 x 3.0 mm id. The mobile phase consisted of a mixture of acetonitrile and buffer (10 mM sodium octane-1-sulfonate, adjusted with H3PO4 to pH 2.2; 200 + 800, v/v), with a constant flow rate of 0.3 mL/min. The separation was carried out at 30 degrees C, and the injection volume was 3 microL. Fluorescence detection was performed at excitation and emission wavelengths of 275 and 310 nm, respectively. The mobile phase parameters, such as the organic solvent fraction (acetonitrile) in mobile phase as an organic modifier, the concentration of sodium octane-1-sulfonate as a counter-ion, temperature, and pH of mobile phase, were studied. As an alternative to ion-pair chromatography, hydrophilic interaction liquid chromatography (HILIC) was investigated using a Luna HILIC column, 3 microm, 100 x 4.6 mm id. The mobile phase consisted of acetonitrile and buffer (5 mM potassium dihydrogen phosphate, adjusted with H3PO4 to pH 2.5; 750 + 250, v/v) at a flow rate of 0.8 mL/min. The separation was carried out at 25 degrees C, and the injection volume was 5 microL. The proposed method has an advantage of a very simple sample pretreatment, and is much faster than the currently utilized HPLC methods using gradient elution and UV detection. Commercial samples of sachets were successfully analyzed by the proposed HPLC method.     

Simple method for the determination of rosiglitazone in human plasma using a commercially available internal standard

To the best of our knowledge, bioanalytical methods to determine rosiglitazone in human plasma reported in literature use internal standards that are not commercially available. Our purpose was to develop a simple method for the determination of rosiglitazone in plasma employing a commercially available internal standard (IS). After the addition of celecoxib (IS), plasma (0.25 mL) samples were extracted into ethyl acetate. The residue after evaporation of the organic layer was dissolved in 750 microL of mobile phase and 50 microL was injected on to HPLC. The separation was achieved using a Hichrom KR 100, 250 x 4.6 mm C(18) with a mobile phase composition potassium dihydrogen phosphate buffer (0.01 m, pH 6.5):acetonitrile:methanol (40:50:10, v/v/v). The flow-rate of the mobile phase was set at 1 mL/min. The column eluate was monitored by fluorescence detector set at an excitation wavelength of 247 nm and emission wavelength of 367 nm. Linear relationships (r(2) > 0.99) were observed between the peak area ratio rosiglitazone to IS vs rosiglitazone concentrations across the concentration range 5-1000 ng/mL. The intra-run precision (%RSD) and accuracy (%Dev) in the measurement of rosiglitazone were <+/-10.69 and <-12.35%, respectively across the QC levels (50-1000 ng/mL). The extraction efficiency was >80% for both rosiglitazone and IS from human plasma. The lower limit of quantitation of the assay was 5 ng/mL. In summary, the methodology for rosiglitazone measurement in plasma was simple, sensitive and employed a commercially available IS.     

Stereoselective determination of hydroxychloro-quine and its major metabolites in human urine by solid-phase microextraction and HPLC

The enantioselective analysis of hydroxychloroquine (HCQ) and its major metabolites was achieved by HPLC and solid-phase microextraction. The chromatographic separation was performed on a Chiralcel OD-H column using hexane/methanol/ethanol (96:2:2, v/v/v) plus 0.2% diethylamine as the mobile phase, at the flow rate of 1.3 mL/min. The main extraction parameters were optimized. The best condition was achieved by the addition of 10% NaCl and 1 mL phosphate buffer 1 mol/L pH 11 to 3 mL human urine. The extraction was conducted for 40 min at 25 degrees C and the desorption time was 3 min using methanol (100%). PDMS-DVB 60 microm fiber was used in this study. The mean recoveries were 9.3, 9.2, and 14.4% for HCQ, desethylhydroxychloroquine (DHCQ), and desethylchloroquine (DCQ), respectively. The method was linear over the range of 50-1000 ng/mL for HCQ enantiomers and over the range of 42-416 ng/mL for DCQ and DHCQ enantiomers. Within-day and between-day precision and accuracy assays for HCQ and its metabolites were lower than 15%. The preliminary 48 h urinary excretion study performed in human urine showed to be stereoselective. The amount of (+)-(S)-enantiomer excreted was higher than its antipode.     

Automated high-performance liquid chromatographic determination of antipyrine and its main metabolites in plasma, saliva and urine, including 4,4'-dihydroxyantipyrine

A rapid, selective and sensitive method was developed for the determination of antipyrine and its main metabolites in plasma, saliva and urine by an automated high-performance liquid chromatographic system. Using a MOS-Hypersil reversed-phase column with a phosphate buffer--acetonitrile mobile phase, baseline separation of antipyrine, its metabolites 3-hydroxymethylantipyrine, norantipyrine and 4-hydroxyantipyrine, and the internal standard, phenacetin, was achieved within 6 min. Factors regarding the accuracy and precision of the method and the stability of phase I metabolites during sample preparation are discussed, taking into account certain drawbacks of previously published methods. Based on the same chromatographic system a method was developed for the assay of 4,4'-dihydroxyantipyrine in urine. This compound is an important metabolite of antipyrine in the rat, representing 12.6 +/- 1.8% of the administered dose (n = 18).     

Determination of ceftriaxone in cerebrospinal fluid by ion-pair liquid chromatography

An ion-pair liquid chromatographic assay was developed and validated for the determination of ceftriaxone in cerebrospinal fluid. Chromatographic separation was achieved on a C18 column (125 x 4 mm, 5 microm) with detection at 270 nm, a 1 mL/min flow rate and a 50 microL loop. The mobile phase consisted of 300 mL acetonitrile, 50 mL 0.1M phosphate buffer (pH 7.4), 3.2 g tetrabutylammonium bromide as the ion-pairing agent, and dilution with distilled deionized water to 1 L. Cephradine was used as the internal standard. The assay was linear for ceftriaxone concentrations of 0.5-50 microg/mL. The coefficients of variation for precision were <4.61%. The accuracy ranged from 96.07 to 102.42%. The detection and quantitation limits were 0.019 and 0.065 microg/mL, respectively. This method was used to quantify ceftriaxone in the cerebrospinal fluid of children with meningitis. The results showed that the method described here is useful for the determination of ceftriaxone in cerebrospinal fluid.