Novel stir bar sorptive extraction methods for environmental and biomedical analysis

Stir bar sorptive extraction (SBSE) is sample preparation technique that involves the extraction and enrichment of organic compounds from a liquid sample. The technique is based on the principle of sorptive extraction. A large amount of extraction phase is coated on a stir bar. An analyte is extracted into the extraction phase, based on its octanol-water partitioning coefficient and the phase ratio. Recently, various methods involving SBSE were developed in order to further facilitate analysis and improve sensitivity. In this review, we focused on the novel methods that involve SBSE with in situ derivatization, SBSE with in situ de-conjugation, thermal desorption (TD) in the multi-shot mode and TD with in tube derivatization method. Those methods were applied successfully to the trace analysis of environmental and biological samples and extremely low detection limits were achieved.     

Stir bar sorptive extraction-thermal desorption-capillary GC-MS for profiling and target component analysis of pharmaceutical drugs in urine

Stir bar sorptive extraction (SBSE) in combination with thermal desorption (TD) on-line coupled to capillary gas chromatography-mass spectrometry (CGC-MS) was applied to the analysis of pharmaceutical drug compounds and metabolites in urine. SBSE implies stirring of the aqueous sample (urine, blood, etc.) with a glass stir bar coated with a thick layer (24 microl) of polydimethylsiloxane (PDMS) for sorptive enrichment of the analytes of interest. In combination with quantitative TD, on-line coupled with CGC-MS, the technique showed to be very versatile and sensitive for the analysis of a wide range of drug substances. Moreover, the relative high enrichment efficiencies of SBSE allow to use mass spectrometric detection (MSD) in the full scan mode. In situ derivatization of polar compounds before SBSE is demonstrated for the analysis of paracetamol and this resulted in both improved chromatographic behavior and higher sensitivity. The quantitative performance of SBSE-TD-CGC-MS is illustrated with the analysis of some barbiturates in urine.     

Sequential-injection determination of traces of disodium phenyl dibenzimidazole tetrasulphonate in urine from users of sunscreens by on-line solid-phase extraction coupled with a fluorimetric detector

A sensitive and selective method to determine disodium phenyl dibenzimidazole tetrasulphonate (PDT) in the urine of sunscreen users, which is suitable for studies on body accumulation/excretion is proposed. On-line solid-phase extraction allows the analyte to be retained and subsequentely eluted, using a strong anion exchange (SAX) microcolumn. Standard addition calibration was carried out with only one standard. The wavelengths of excitation and emission were 330 and 454 nm, respectively. The method allows PDT to be determined in both, spiked and unspiked human urine samples, without any pre-treatment. Results obtained for spiked urine samples (40-200 ng ml(-1)) showed the accuracy of the method. The mean relative standard deviations (R.S.D.) of the results was 7%. Five volunteers applied a sunscreen lotion containing 5% PDT and their urinary excretion was controlled from the moment of application until the excreted amounts were no longer detectable. The sensitivity of the proposed method is in the order of 1900 ml microg(-1) and the detection limit (3S(y/x)/b) is in the order of 5 ng of PDT, which means 10 ng ml(-1) for a 500 microl injected volume, and this is suitable for the PDT levels found in the urine.     

Simultaneous determination of methamphetamine and amphetamine in human urine using pipette tip solid-phase extraction and gas chromatography-mass spectrometry

Methamphetamine and amphetamine were extracted from human urine samples using pipette tip solid-phase extraction (SPE) with MonoTip C18 tips (pipette tip volume, 200 microl), in which C18-bonded monolithic silica gel was fixed. A sample of human urine (0.5 ml) containing methamphetamine, amphetamine, and N-methylbenzylamine as internal standard (IS), was mixed with 25 microl of 1M sodium hydroxide solution. The mixture was extracted into the C18 phase of the SPE tip by 25 repeated aspirating/dispensing cycles using a manual micropipettor. Analytes retained in the C18 phase were then eluted with methanol by five repeated aspirating/dispensing cycles. After derivatization with trifluoroacetic anhydride, analytes were measured by gas chromatography/mass spectrometry with selected ion monitoring in the positive-ion electron impact mode. Recoveries of methamphetamine, amphetamine, and IS spiked into urine were more than 82.9, 82.2, and 78.2%, respectively. Regression equations for methamphetamine and amphetamine showed excellent linearity in the range of 0.25-200 ng/0.5 ml. Limit of detection was 0.04 ng/0.5 ml for methamphetamine and 0.05 ng/0.5 ml for amphetamine. Intra- and inter-day coefficients of variations for both stimulants were not greater than 10.8%. The data obtained from actual determination of methamphetamine and amphetamine in autopsy urine samples are also presented for validation of the method.     

Determination and assay validation of luteolin and apigenin in human urine after oral administration of tablet of Chrysanthemum morifolium extract by HPLC

A simple, selective, precise, and accurate RP-HPLC assay for simultaneous analysis of luteolin and apigenin in human urine was developed and validated. Prior to HPLC analysis, urine samples were incubated with beta-glucuronidase/sulfatase. Separation and quantification were achieved on an Agilent C18 column under isocratic conditions using a mobile phase (methanol:0.2% phosphoric acid aqueous solution 55:45, v/v) maintained at 1.0 ml/min at 30 degrees C. The standard curves were linear over the range of 0.0975-7.800 and 0.1744-13.95 microg/ml for luteolin and apigenin, respectively (r > 0.999). The assay recoveries for luteolin and apigenin were above 85.7%. The intra-day and inter-day precision (R.S.D.) for luteolin were below 2.2 and 4.0%, respectively, and for apigenin were less than 2.8 and 5.4%, respectively. Stability studies showed three concentration of luteolin and apigenin in urine quality control samples were stable undergoing three freeze-thaw cycles, storage at room temperature for 4 h, and at -20 degrees C for 3 days. The limit of quantitation was 39.20 ng/ml (n = 5) for luteolin and 31.45 ng/ml (n = 5) for apigenin in human urine. The method developed was employed successfully to determine luteolin and apigenin in urine samples obtained from eight healthy volunteers following oral administration of tablet of Chrysanthemum morifolium extract (CME).     

Determination of a new phosphodiesterase V inhibitor,DA-8159, in plasma and urine by high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) method using liquid-liquid extraction for sample preparation was developed for the determination of a new phosphodiesterase V inhibitor, DA-8159, in rat plasma and urine using sildenafil citrate as an internal standard. A 100 microl aliquot of 0.1 M Na(2)CO(3) (containing sildenafil citrate, 3 microg/ml as free sildenafil) and a 1 ml aliquot of ether were added to a 100 microl aliquot of biological samples (urine samples were diluted 20 times with distilled water). After vortex centrifugation at 9000 x g for 3 min, the ether layer was collected and dried under nitrogen gas. The residue was reconstituted with a 150 microl aliquot of the mobile phase, centrifuged, and a 100 microl aliquot of the supernatant was injected onto a reversed-phase column. The mobile phases, 20 mM KH(2)PO(4) (pH 4.7):acetonitrile (70:30, v/v for plasma and tissue samples, and 75:25, v/v for urine samples), were run at a flow rate of 1.0 ml/min. The column effluent was monitored by an ultraviolet detector set at 292 nm. The retention times for DA-8159 and the internal standard were approximately 10.7 and 9.1 min, respectively, in plasma and tissue samples and the corresponding values in urine samples were 47 and 33 min. The detection limits for DA-8159 in rat plasma and urine were 20 and 100 ng/ml, respectively. The coefficients of variation of the assay were generally low: below 10% for plasma and 9.9% for urine. No interferences from endogenous substances were found.     

Stir bar sorptive extraction of diclofenac from liquid formulations: a proof of concept study

A new stir bar sorptive extraction (SBSE) technique coupled with HPLC-UV method for quantification of diclofenac in pharmaceutical formulations has been developed and validated as a proof of concept study. Commercially available polydimethylsiloxane stir bars (Twister™) were used for method development and SBSE extraction (pH, phase ratio, stirring speed, temperature, ionic strength and time) and liquid desorption (solvents, desorption method, stirring time etc) procedures were optimised. The method was validated as per ICH guidelines and was successfully applied for the estimation of diclofenac from three liquid formulations viz. Voltarol^® Optha single dose eye drops, Voltarol^® Ophtha multidose eye drops and Voltarol^® ampoules. The developed method was found to be linear (r = 0.9999) over 100–2000 ng/ml concentration range with acceptable accuracy and precision (tested over three QC concentrations). The SBSE extraction recovery of the diclofenac was found to be 70% and the LOD and LOQ of the validated method were found to be 16.06 and 48.68 ng/ml, respectively. Furthermore, a forced degradation study of a diclofenac formulation leading to the formation of structurally similar cyclic impurity (indolinone) was carried out. The developed extraction method showed comparable results to that of the reference method, i.e. method was capable of selectively extracting the indolinone and diclofenac from the liquid matrix. Data on inter and intra stir bar accuracy and precision further confirmed robustness of the method, supporting the multiple re-use of the stir bars.     

Quantification of carbamazepine, carbamazepine-10,11-epoxide, phenytoin and phenobarbital in plasma samples by stir bar-sorptive extraction and liquid chromatography

A sensitive and reproducible stir bar-sorptive extraction and high-performance liquid chromatography-UV detection (SBSE/HPLC-UV) method for therapeutic drug monitoring of carbamazepine, carbamazepine-10,11-epoxide, phenytoin and phenobarbital in plasma samples is described and compared with a liquid:liquid extraction (LLE/HPLC-UV) method. Important factors in the optimization of SBSE efficiency such as pH, extraction time and desorption conditions (solvents, mode magnetic stir, mode ultrasonic stir, time and number of steps) assured recoveries ranging from 72 to 86%, except for phenytoin (62%). Separation was obtained using a reverse phase C(18) column with UV detection (210nm). The mobile phase consisted of water:acetonitrile (78:22, v/v). The SBSE/HPLC-UV method was linear over a working range of 0.08-40.0mugmL(-1) for carbamazepine, carbamazepine-10,11-epoxide and phenobarbital and 0.125-40.0mugmL(-1) for phenytoin, The intra-assay and inter-assay precision and accuracy were studied at three concentrations (1.0, 4.0 and 20.0mugmL(-1)). The intra-assay coefficients of variation (CVs) for all compounds were less than 8.8% and all inter-CVs were less than 10%. Limits of quantification were 0.08mugmL(-1) for carbamazepine, carbamazepine-10,11-epoxide and phenobarbital and 0.125mugmL(-1) for phenytoin. No interference of the drugs normally associated with antiepileptic drugs was observed. Based on figures of merit results, the SBSE/HPLC-UV proved adequate for antiepileptic drugs analyses from therapeutic levels. This method was successfully applied to the analysis of real samples and was as effective as the LLE/HPLC-UV method.     

Determination of steroid sex hormones in water and urine matrices by stir bar sorptive extraction and liquid chromatography with diode array detection

In this study, stir bar sorptive extraction and liquid desorption followed by high performance liquid chromatography with diode array detection (SBSE-LD-HPLC/DAD) were combined for the simultaneous determination of nine steroid sex hormones (estrone, 17alpha-estradiol, 17beta-estradiol, 17alpha-ethynylestradiol, diethylstilbestrol, mestranol, progesterone, 19-norethisterone and norgestrel) in water and urine matrices. During the method development, it has been demonstrated that equilibrium time, ionic strength and back extraction solvents are the most important parameters to control, for determining the nine-hormones in water matrices, in which stir bars coated with 126 microl of polydimethylsiloxane were used. Assays performed on 30 ml water samples spiked at 10 microg/l levels under optimised experimental conditions, yielded recoveries ranging from 11.1+/-4.9% (17beta-estradiol) to 100.2+/-10.4% (mestranol), showed that the methodology is well described by the octanol-water partition coefficients (K(PDMS/W) approximately K(O/W)) for the latter, while pronounced deviations to the theoretical efficiency (K(PDMS/W) not equal K(O/W)) were observed for the remaining hormones. From calibration studies, a good analytical performance for all hormones was attained, including a suitable precision (2.1-17.1%), low limits of detection (0.3-1.0 microg/l) and an excellent linear dynamic range (1.25-50.0 microg/l). Assays on environmental water and urine matrices showed recovery yields in worthy good agreement with the spiking level (10 microg/l), and suitability for profiling low microg/l levels of natural hormones in urine samples taken from pregnant women. The present methodology is easy, reliable and sensitive at the trace level, only requiring a low sample volume, showing to be a good analytical alternative to routine quality control for environmental and biomedical laboratories.     

Sensitive SPE-HPLC method to determine a novel angiotensin-AT1 antagonist in biological samples

A high-performance liquid chromatography (HPLC)-method after solid-phase extraction (SPE) has been developed in order to determine a new angiotensin-AT1 antagonist, i.e. CR 3210 (C27H24N8; MW = 460.54), 4-[4-[(2-ethyl-5,7-dimethylimidazo[4,5-b]pyridin-3-yl)methyl]phenyl]-3-(2H-tetrazol-5-yl)quinoline in rat plasma and urine after oral administration to Sprague-Dawley rats. CR 3210 and the internal standard (IS) CR 1505 (loxiglumide), i.e. 4-[(3,4-dichlorobenzoyl)amino]-5-[(3-methoxypropyl)pentylamino]-5-oxopentanoic acid, were isolated from rat urine and plasma by solid-phase extraction. The procedure was optimized regarding the sorbent extraction material, the pH in the conditioning solution, the washing step, the dry time and the type of elution solvent. The separation was performed by reversed-phase high-performance liquid chromatography with ultraviolet detection. The samples were injected onto the analytical column (Tracer Extrasil ODS1) and detected at 238 nm, giving a capacity factor of 1.87 for CR 3210 and 1.10 for the internal standard. The selectivity of the method was satisfactory. The mean recovery of CR 3210 from spiked rat plasma was 68.5 at 75 ng/ml and 80.9 at 3000 ng/ml; the mean recovery of CR 3210 from spiked rat urine was 69.9 at 75 ng/ml and 78.6 at 3000 ng/ml. The lower limit of detection (LOD) was 14 ng/ml in plasma and 22 ng/ml in urine samples. The lower limit of quantification (LOQ) was taken as 30 ng/ml, the lowest calibration standard using 500 microl rat plasma and urine. The procedures were validated according to international standards with a good reproducibility and linear response from 30 to 3000 ng/ml, for either plasma or urine. The sensitivity of the method allowed for its application to pharmacokinetic studies.     

Development of a stir bar sorptive extraction based HPLC-FLD method for the quantification of serotonin reuptake inhibitors in plasma,urine and brain tissue samples

The aim of this article is to present an analytical application of stir bar sorptive extraction (SBSE) coupled to HPLC-fluorescence detection (FLD) for the quantification of fluoxetine (FLX), citalopram (CIT) and venlafaxine (VLF) and their active metabolites – norfluoxetine (NFLX), desmethyl- (DCIT) and didesmethylcitalopram (DDCIT) and o-desmethylvenlafaxine (ODV) – in plasma, urine and brain tissue samples. All the parameters influencing adsorption (pH, ion strength, organic modifier addition, volume, extraction time and temperature) and desorption (desorption solvent composition, time, temperature and desorption mode) of the analytes on the stir bar have been optimized. For each matrix, the analytical method has been assessed by studying the linearity and the intra- and interday accuracy (89–113%) and precision (RSD < 13%). The improvement of the quantification limits (0.2–2 μg l⁻¹ for plasma, 2–20 ng g⁻¹ for brain tissue and 1–10 μg l⁻¹ for urine, depending on the respective response for analytes) and the development of a procedure for all the matrices make this method useful in clinical and forensic analysis.     

Formation of the quaternary ammonium-linked glucuronide of nicotine in human liver microsomes: identification and stereoselectivity in the kinetics.

The formation of the N1-glucuronide metabolite of each nicotine enantiomer was studied in pooled human liver microsomes (n = 6). The metabolite formed from natural S(-)-nicotine was identified by comparison of the high-pressure liquid chromatography (HPLC) retention time and positive ion electrospray ionization-mass spectral characteristics with a synthetic reference standard. A radiometric HPLC method was used to quantify the metabolite. The specificity of the assay method was demonstrated by experiments in which beta-glucuronidase treatment of incubated assay samples resulted in elimination of the peak due to the N1-glucuronide metabolite. The glucuronides of S(-)- and R(+)-nicotine were formed by one-enzyme kinetics, with K(m) values of 0.11 and 0.23 mM and V(max) values of 132 and 70 pmol/min/mg of protein, respectively. There is marked stereoselectivity in the apparent intrinsic clearance values (V(max)/K(m)) in that the value for S(-)-nicotine is 4 times greater than for the R(+)-isomer (1.2 versus 0.31 microl/min/mg of protein).     

Method of simultaneous stir bar sorptive extraction of phenethylamines and THC metabolite from urine

Stir bar sorptive extraction is a technique used for extracting target substances from various aqueous matrixes such as environmental water, food, and biological samples. This type of extraction is carried out by rotating a coated stir bar is rotated in the sample solution. In particular, Twister bar is a commercial stir bar that is coated with polydimethylsiloxane (PDMS) and used to perform sorptive extraction. In this study, we developed a method for simultaneous detection of amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxymethamphetamine, and a Δ(9)-tetrahydrocannabiniol (THC) metabolite in human urine. For extracting the target analytes, the Twister bar was simply stirred in the sample in the presence of a derivatizing agent. Using this technique, phenethylamines and the acidic THC metabolite can be simultaneously extracted from human urine. This method also enables the extraction of trace amounts of these substances with good reproducibility and high selectivity. The proposed method offers many advantages over other extraction-based approaches and is therefore well suited for screening psychoactive substances in urine specimens.     

Determination of oxprenolol and its glucuronide metabolite in plasma and urine using high-performance liquid chromatography

A HPLC assay is presented for the determination of oxprenolol (1) and its glucuronic acid conjugate (2) in human plasma and urine. The procedure employs a selective re-extraction using alprenolol (3) as the internal standard, followed by reversed-phase chromatography and UV-detection. The minimal detectable concentration is 10 ng/ml in plasma and 50 ng/ml in urine, using 1.0 and 0.5 ml of plasma and urine, respectively. Within-run and day-to-day variations are below 10% at all concentrations examined. Plasma and urine samples of either healthy volunteers or patients with renal failure are free of interferences from endogenous compounds and drugs frequently used in these patients. The glucuronic acid conjugate of oxprenolol is determined as the parent compound after hydrolytic cleavage with beta-glucuronidase/arylsulfatase. The specificity and selectivity of this cleavage are also demonstrated.     

Improved RP-HPLC method to determine biapenem in human plasma/urine and its application to a pharmacokinetic study

Existing methods to determine biapenem (CAS 120410-24-4), a carbapenem, either lacked sensitivity/reproducibility or had no internal standard as a control. Here an improved reversed-phase high-performance liquid chromatographic (RP-HPLC) method was established in human plasma and urine. After adding p-aminobenzoic acid as the internal standard to plasma or urine, plasma samples were ultra-filtrated and urine samples were diluted directly. Chromatographic separations were carried out on a 4.6 mm x 150 mm column with acetonitrile-0.1 mol/l sodium acetate (2:98, v:v; pH 4.38 or 4.00) as mobile phase and UV detection at 300 nm. The extraction recovery was 91.51% for biapenem at the concentration level of 5 microg /ml in human plasma. The linear quantification range of the method was 0.1 to approximately 50 microg /ml for plasma and urine, with linear correlation coefficients greater than 0.998. The intra-day and inter-day relative standard deviations (R.S.D.) for biapenem at low, middle and high levels in human samples were less than 12.51% for plasma and less than 7.05% for urine. The RP-HPLC method was successfully applied to pharmacokinetic studies, in which healthy subjects received multiple doses of biapenem (300 mg, i.v., b.i.d., for 5 continuous days). The pharmacokinetic results are presented.     

Inhibition of phenol sulfotransferase (SULT1A1) by quercetin in human adult and foetal livers

1. Quercetin is one of the most abundant flavonoids in edible vegetables, fruit and wine. The aim was to study the type of inhibition of SULT1A1 by quercetin in the human adult and foetal livers. 2. The activity of SULT1A1 was measured with 4 microM 4-nitrophenol and 0.4 microM 3'-phosphoadenosine-5'-phosphosulphate-[(35)S], and its mean (+/-SD) and median were 769 +/- 311 and 740 pmol min(-1) mg(-1), respectively (adult liver, n = 10), and 185 +/- 98 and 201 pmol min(-1) mg(-1), respectively (foetal liver, n = 8, p < 0.0001). 3. In non-inhibited samples, K(m) for SULT1A1 (mean +/- SD) was 0.31 +/- 0.14 microM (adult liver) and 0.49 +/- 0.17 microM (foetal liver, n.s.). V(max) for SULT1A1 (mean +/- SD) was 885 +/- 135 pmol min(-1) mg(-1) (adult liver) and 267 +/- 93 pmol min(-1) mg(-1) (foetal liver, p = 0.007). 4. The IC(50) of quercetin for SULT1A1 was measured in three samples of adult and foetal livers and was 13 +/- 2.1 and 12 +/- 1.4 nM, respectively. 5. The type of inhibition was mixed non-competitive in adult and foetal livers and K(i) was 4.7 +/- 2.5 nM (adult liver) and 4.8 +/- 1.6 nM (foetal liver). 6. In the adult liver, the intrinsic clearance (mean +/- SD) was 3.3 +/- 1.5 ml min(-1) mg(-1) (non-inhibited samples), 0.9 +/- 0.4 ml min(-1) mg(-1) (12.5 nM quercetin) and 0.5 +/- 0.06 ml min(-1) mg(-1) (25 nM quercetin). In the foetal liver, the intrinsic clearance (mean +/- SD) was 0.5 +/- 0.2 ml min(-1) mg(-1) (non-inhibited samples), 0.12 +/- 0.01 ml min(-1) mg(-1) (12.5 nM quercetin) and 0.2 +/- 0.09 ml min(-1) mg(-1) (25 nM quercetin). 7. In conclusion, quercetin is a potent inhibitor of human adult and foetal liver SULT1A1. It reduces the sulphation rate and intrinsic clearance of 4-nitrophenol in both human adult and foetal livers. This suggests that quercetin may inhibit the sulfation rate of those drugs sulphated by SULT1A1. The inhibition of SULT1A1 is complex and not due solely to competition at the catalytic site of SULT1A1.     

A sensitive fluorescence optosensor for analysing propranolol in pharmaceutical preparations and a test for its control in urine in sport

We describe a simple and selective method for analysing propranolol and a sensitive test for its control in urine. A flow-through fluorescence optosensor based on on-line immobilization in a non-ionic-exchanger (Amberlite XAD-7) solid support in a continuous flow was used in both cases. Determination was made in 5 mM H(2)PO(4)(-)/HPO(4)(2-) buffer solution at pH 6 at a working temperature of 20 degrees C. Fluorescence intensities were measured at lambda(ex/em) = 300/338 nm with a response time of 80 s, thus obtaining a linear concentration range of between 0 and 250.0 ng ml(-1) with a detection limit of 1.3 ng ml(-1), an analytical sensitivity of 6.0 ng ml(-1) and a standard deviation of 2.40% at a 150 ng ml(-1) concentration level for propranolol. We also propose a test to detect propranolol in urine with a linear concentration range between 0 and 100.0 ng ml(-1), a detection limit of 0.2 ng ml(-1), an analytical sensitivity of 1.0 ng ml(-1), and a standard deviation of 0.84% at a 75 ng ml(-1) concentration level. The effect of proteins presents in urine samples were evaluated. The two proposed methods were satisfactorily applied to commercial formulations and urine samples respectively.     

High performance liquid chromatographic analysis of a novel aminoalkylpyridine anticonvulsant 2-(4-chlorophenyl)amino-2-(4-pyridyl)ethane

A simple, rapid and specific high performance liquid chromatographic (HPLC) method for the quantitation of 2-(4-chlorophenyl)amino-2-(4-pyridyl)ethane (AAP-Cl) and identification of its putative metabolite, 2-(4-chlorophenyl)amino-2-(4-pyridyl)ethanol (beta-AA) in rat blood and urine has been developed. AAP-Cl, beta-AA and an appropriate internal standard were extracted from rat biofluids by a solid phase extraction technique using C18 cartridges prior to the HPLC analysis. The extractibility was 92% for AAP-Cl and 98% for beta-AA. The HPLC analysis employed a symmetrical or standard reversed-phase HPLC column (Apex ODS, 5 microm, 25 cm x 0.46 cm) for blood or urine analysis, a mobile phase of water methanol acetonitrile (40:30:30) containing 20 microl 100 ml(-1) diethylamine at a flow rate of 1 ml min(-1), and UV detection at 254 nm. The limit of detection was 100 ng ml(-1) for both analytes in both blood and urine. The calibration curves for AAP-Cl in rat biofluids were shown to be linear in both low and high concentration ranges (blood: 0-1 and 1-10 microg ml(-1); urine: 0-10 and 10-100 microg ml(-1)) with intra- and inter-day coefficients of variation of no more than 18% for blood and 14% for urine. The method developed was successfully applied to a preliminary analysis of intact AAP-Cl in both blood and urine obtained from rats dosed with AAP-Cl.     

Determination of 3'-C-ethynylcytidine in human plasma and urine by liquid chromatographic-electrospray ionization tandem mass spectrometry

A liquid chromatographic-electrospray ionization tandem mass spectrometric (LC-ESI/MS/MS) method was developed for the quantitative analysis of a novel anticancer drug, 3'-C-ethynylcytidine (I) in human plasma and urine. I and its stable isotope-labeled internal standard (II) were extracted from human plasma and urine samples using a polymer-based cation-exchange cartridge, and LC-ESI/MS/MS analysis was performed by monitoring the positive fragment ions of I and II. The linear ranges are 1-500 ng/ml in plasma and 10-5000 ng/ml in urine. The limits of quantitation for I were 1 ng/ml in plasma and 10 ng/ml in urine. The relative errors (RE) for I ranged from -8.4 to 3.0% in plasma and from 0.8 to 4.4% in urine. The relative standard deviations (RSD) for I ranged from 1.2 to 8.9% in plasma and from 0.7 to 2.8% in urine. This validated analytical method is demonstrated to be useful for the analysis of I in human plasma and urine in clinical studies.