Quantification of cidofovir in human serum by LC-MS/MS for children

A new method for the quantification of cidofovir (CDV), an acyclic nucleotide analogue of cytosine with antiviral activity against a broad-spectrum of DNA viruses, in human serum, using high-performance liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) has been developed. A strong anion exchange (SAX) solid-phase extraction procedure was applied for the sample preparation. The tandem mass spectrometer was tuned in the multiple reaction monitoring mode to monitor the m/z 278.1-->234.9 and the m/z 288.1-->133.1 transitions for CDV and the internal standard 9-(2-phosphonylmethoxyethyl)guanine (PMEG), respectively, using negative electrospray ionization. The MS/MS response was linear over the concentration range from 78.125 ng/ml to 10,000 ng/ml, with a lower limit of quantification of 78.125 ng/ml. The intra- and inter-day precisions (relative standard deviation (%)) for CDV were less than 7.8% and the accuracies (% of deviation from nominal level) were within +/-12.1% for quality controls. The novel LC-MS/MS method allowed a specific, sensitive and reliable determination of CDV in human serum and was applied to investigate the yet unknown pharmacokinetic properties of CDV in a paediatric cancer patient.     

Liquid chromatography-tandem mass spectrometry validated method for the estimation of indapamide in human whole blood

A highly precise and sensitive method for the estimation of indapamide in human whole blood using high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) is described. The method developed is validated in human whole-blood matrix, with a sensitivity of 0.5 ng/ml as lower limit of quantification. The procedure for the extraction of indapamide and glimepiride as internal standard (IS) involves haemolysis and deprotienation of whole blood using ZnSO(4) followed by liquid-liquid extraction using ethyl acetate. The sample extracts after drying were reconstituted and analysed by LC-MS/MS, equipped with turbo ion spray (TIS) source, operating in the positive ion and selective reaction monitoring (SRM) acquisition mode to quantify indapamide in human whole blood. The mean recovery for indapamide was 82.40 and 93.23% for IS. The total run time was 2.5 min to monitor both indapamide and the IS. The response of the LC-MS/MS method for indapamide was linear over the range of 0.5-80.0 ng/ml with correlation coefficient, r>or=0.9991. The coefficient of variance (% CV) at 0.5 ng/ml was 4.02% and the accuracy was well within the accepted limit of +/-20% at 0.5 ng/ml and +/-15% at all other concentrations in the linear range. This method is fully validated for the accuracy, precision and stability studies and also applied to subject-sample analysis of bioequivalence study for 1.5mg sustained-release (SR) formulations.     

Determination of amiodarone and desethylamiodarone in horse plasma and urine by high-performance liquid chromatography combined with UV detection and electrospray ionization mass spectrometry

A rapid method for the quantification of amiodarone and desethylamiodarone in animal plasma using high-performance liquid chromatography combined with UV detection (HPLC-UV) is presented. The sample preparation includes a simple deproteinisation step with acetonitrile. In addition, a sensitive method for the quantification of amiodarone and desethylamiodarone in horse plasma and urine using high-performance liquid chromatography combined with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) is described. The sample preparation includes a solid-phase extraction (SPE) with a SCX column. Tamoxifen is used as an internal standard for both chromatographic methods. Chromatographic separation is achieved on an ODS Hypersil column using isocratic elution with 0.01% diethylamine and acetonitrile as mobile phase for the HPLC-UV method and with 0.1% formic acid and acetonitrile as mobile phase for the LC-MS/MS method. For the HPLC-UV method, good linearity was observed in the range 0-5 microg ml(-1), and in the range 0-1 microg ml(-1) for the LC-MS/MS method. The limit of quantification (LOQ) was set at 50 and 5 ng ml(-1) for the HPLC-UV method and the LC-MS/MS method, respectively. For the UV method, the limit of detection (LOD) was 15 and 10 ng ml(-1) for amiodarone and desethylamiodarone, respectively. The LODs of the LC-MS/MS method in plasma were much lower, i.e. 0.10 and 0.04 ng ml(-1) for amiodarone and desethylamiodarone, respectively. The LODs obtained for the urine samples were 0.16 and 0.09 ng ml(-1) for amiodarone and desethylamiodarone, respectively. The methods were shown to be of use in horses. The rapid HPLC-UV method was used for therapeutic drug monitoring after amiodarone treatment, while the LC-MS/MS method showed its applicability for single dose pharmacokinetic studies.     

Determination of vancomycin in serum by liquid chromatography-high resolution full scan mass spectrometry

A liquid chromatography-mass spectrometry (LC-MS) method was developed for the analysis of vancomycin (VCM) in human serum. The method was based on full scan data with extracted ions for the accurate masses of VCM and the atenolol internal standard obtained by Fourier transform MS. VCM was extracted from serum using strong cation exchange (SCX) solid phase extraction (SPE). The method was found to be linear in the range 0.05-10 microg/ml, which was adequate for quantification of VCM in serum samples, with a limit of quantification (LOQ) of 0.005 microg/ml and a limit of detection (LOD) of 0.001 microg/ml. Intra-day precision (n=5) was +/-3.5%, +/-2.5%, +/-0.7% at 0.05, 0.5 and 5 microg/ml, respectively. Inter-day precision (n=5) was +/-7.6%, +/-6.4%, +/-3.9% at 0.05, 0.5 and 5 microg/ml, respectively. The process efficiency for VCM was in the range 89.2-98.1% with the recovery for the atenolol internal standard (IS) being 97.3%. The method was used to determine VCM levels in patients during peri-operative infusion of the drug, which was found to result in drug levels within the required therapeutic window.     

Determination of captopril in human plasma, using solid phase extraction and high-performance liquid chromatography, coupled to mass spectrometry: application to bioequivalence study

A specific high performance liquid chromatography-mass spectrometric (LC-MS/MS) assay was developed for the determination of captopryl in plasma. The retention time was 1.45 and 1.37 min for captopril and enalapril, respectively. The overall mean recovery, using SPE extraction with OASIS HLB cartridges, was found to be 107.2+/-9.5 and 100.04+/-2%, respectively. Calibration curves were linear in the concentration range of 10.00-2000.00 ng/ml, and the lower limit of quantification (LLOQ) was 10.00 ng/ml. The LLOQ was sensitive enough for detecting terminal phase concentrations of the drug. Inter-batch precision of the method ranged from 0.88 to 1.95%. Intra-batch accuracy ranged from 97.15 to 105.77%, while intra-batch precision ranged from 2.49 to 5.66% at concentrations of 30.00, 760.00 and 1500.00 ng/ml. The developed method was applied to study bioequivalence of captopril in a group of 25 human subjects at a single oral dose of a 50mg tablet.     

Determination of testosterone in saliva and blow of bottlenose dolphins (Tursiops truncatus) using liquid chromatography-mass spectrometry

A rapid, accurate and reproducible assay utilising high performance liquid chromatography-mass spectrometry (LC-MS) has been developed and validated for determining testosterone concentrations in saliva and blow of bottlenose dolphins. Sample preparation used solid phase extraction with specific preconditioning of cartridges. Analytes were eluted with 100% acetonitrile, dried under nitrogen and stored at -80 degrees C. Samples were reconstituted in 60% acetonitrile for LC-MS analysis. Chromatographic separation was achieved with an Alltech Macrosphere C8 stainless steel analytical column (2.1 mm x 150 mm i.d., 5 microm particle size, 300 angstroms pore size) using a 55% mobile phase B isocratic method (mobile phase A = 0.5% acetic acid; mobile phase B = 0.5% acetic acid, 90% acetonitrile). Samples were analysed in SIM at m/z 289.20 (testosterone mw 288.40) and a positive ion ESI. The limit of quantification was 0.5 ng/ml with a limit of detection of 0.2 ng/ml. The concentration curve was linear from 0.5 to 50 ng/ml (y = 0.01x + 0.0045, r(2) = 0.959, r = 0.979, p < 0.001). The R.S.D.s of intra- and inter-batch precision were less than 15% for saliva and 11% blow. Recovery of the assay for saliva was 93.0 +/- 7.9% (50 ng/ml) and 91.5 +/- 3.72% (1 ng/ml), and for blow was 83.3 +/- 6.8% (50 ng/ml) and 85.8 +/- 4.6% (1 ng/ml). Recovery of the internal standard in saliva was 73.0 +/- 14.2% and in blow was 78.63 +/- 4.29. The described assay was used to determine the presence of endogenous testosterone in saliva (9.73-23 ng/ml, n = 10) and blow (14.71-86.20 ng/ml, n = 11) samples of captive bottlenose dolphins.     

Highly sensitive determination of estrone and estradiol in human serum by liquid chromatography-electrospray ionization tandem mass spectrometry

A highly sensitive and specific quantification method of estrone and estradiol in human serum was described based upon the use of picolinoyl derivatization and liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) in a positive mode. Estrogens were treated with picolinoyl chloride hydrochloride or picolinic acid and 2-methyl-6-nitrobenzoic anhydride followed by a solid-phase extraction with ODS cartridge. Picolinoyl derivatization proceeded quantitatively even in a microscale, and the picolinoyl esters provided simple positive ESI-mass spectra showing [M+H](+) as base peaks for these estrogens. The picolinoyl derivatives of these estrogens showed 100-fold higher detection response compared to underivatized intact molecules by LC-ESI-MS (selected reaction monitoring). Using this derivatization, estrogens spiked in the charcoal treated human serum samples were analyzed with limit of quantification (LOQ), intra-day accuracy and precision of 1.0pg/ml, 96.0% and 9.9% for estrone, and 0.5pg/ml, 84.4% and 12.8% for estradiol, respectively. Estrone and estradiol added to the crude serum samples were recovered with comparable LOQ and accuracy obtained for the charcoal treated serum samples as well.     

Ultra sensitive method for the determination of 9-(2-phosphonylmethoxyethyl)adenine in human serum by liquid chromatography-tandem mass spectrometry

An ultra sensitive method for the direct measurement of 9-(2-phosphonylmethoxyethyl)adenine (PMEA), an antiviral agent for hepatitis B, in human serum using high performance liquid chromatography/tandem mass spectrometry (LC-MS/MS) has been developed. This method involves the addition of [13C]PMEA (contains 5 13C) as internal standard, the purification and enrichment by a MCX solid phase extraction (SPE) cartridge, and quantitative analysis using LC-MS/MS. The MS/MS is selected to monitor the m/z 272 --> 134 and m/z 277 --> m/z 139 transitions for PMEA and [13C]PMEA, respectively, using negative electrospray ionization. The MS/MS response is linear over a concentration of 0.1-10 ng/ml with a lower limit of quantitation (LLOQ) of 0.1 ng/ml. The mean inter-assay accuracy (%Bias) for quality control (QC) at 0.1, 0.25, 1.0, and 10 ng/ml are 10, 1.6, -0.8, and 0.0%, respectively. The mean inter-assay precision (%CV) for the corresponding QCs is 3.9, 3.8, 5.3, and 3.4%, respectively. The method has been used to determine PMEA concentration in human serum following a single oral administration of a PMEA pro-drug at dose of 10 and 30 mg.     

Determination of 1,2-bis (2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA free acid) in rat plasma, urine and feces by liquid chromatography with UV and tandem mass spectrometric detection

BAPTA free acid was identified as the main metabolic product of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(actoxymethyl ester) (BAPTA-AM), a neuroprotective agent in cerebral ischemia, in rats. In this paper, liquid chromatography-ultraviolet (LC-UV) and mass spectrometry/mass spectrometry (LC-MS/MS) methods were employed for the determination of BAPTA free acid in rat urine and feces and rat plasma, respectively. By liquid-liquid extraction and LC-UV analysis, a limit of quantitation of 1000 ng/ml using 0.2 ml rat urine for extraction and 250 ng/ml using 1 ml rat fecal homogenate supernatant for extraction could be reached. The assay was linear in the range of 1000-50,000 ng/ml for rat urine and 250-10,000 ng/ml for rat fecal homogenate supernatant. Because the sensitivity of the LC-UV method was apparently insufficient for evaluating the pharmacokinetic profile of BAPTA in rat plasma, a LC-MS/MS method was subsequently developed for the analysis of BAPTA free acid. By protein precipitation and LC-MS/MS analysis, the limit of quantitation was 5 ng/ml using 0.1 ml rat plasma and the linear range was 5.0-500 ng/ml. Both methods were validated and can be used to support a thorough preclinical pharmacokinetic evaluation of BAPTA-AM liposome injection.     

Automated analysis of quetiapine and other antipsychotic drugs in human blood by high performance-liquid chromatography with column-switching and spectrophotometric detection

An automated HPLC method with column switching is described for the determination of quetiapine, clozapine, perazine, olanzapine and metabolites in blood serum. After clean-up on silica C8 material (20 microm particle size) drugs were separated on ODS Hypersil C18 material (5 microm; column size 250 mm x 4.6 mm i.d.) within 25 min and quantified by ultraviolet (UV) detection at 254 nm. The limit of quantification ranged between 10 and 50 ng/ml. At therapeutic concentrations of the drugs, the inter-assay reproducibility was below 10%. Analyses of drug concentrations in serum of 75-295 patients treated with therapeutic doses of the antipsychotic drugs revealed mean+/-S.D. steady state concentrations of 139+/-136 ng/ml for quetiapine, 328+/-195 ng/ml for clozapine, 48+/-27 ng/ml for olanzapine and 71+/-52 ng/ml for perazine. The method was thus suitable for routine therapeutic drug monitoring and may be extended to other drugs.     

Sensitive isotope dilution liquid chromatography/tandem mass spectrometry method for quantitative analysis of bumetanide in serum and brain tissue

We have developed and validated a simple and sensitive stable isotope dilution liquid chromatography/tandem mass spectrometric (LC-MS/MS) method for the quantification of bumetanide in human serum. Samples were prepared with a simple acetonitrile based protein precipitation. The supernatant was then analyzed directly using LC-MS/MS. Chromatographic separation was achieved on a C18 reversed phase column using a methanol and water gradient. The detection was performed in selected reaction monitoring (SRM) mode via a positive electrospray ionization (ESI) interface. The method had a lower limit of quantification (LLOQ) of 1 ng/mL, linearity up to 1250 ng/mL, intra- and inter-day precision less than 10%, and accuracy within ±10%. This method was also demonstrated to be suitable for the analysis of bumetanide in rat serum and brain tissue. Bumetanide concentrations in rat serum and brain were determined for samples collected at several intervals following intraperitoneal (i.p.) injection of bumetanide, and were used to calculate bumetanide permeability through the blood-brain barrier.     

Highly sensitive quantification of serum malonate, a possible marker for de novo lipogenesis, by LC-ESI-MS/MS

We describe a new sensitive and specific method for the quantification of serum malonate (malonic acid, MA), which could be a new biomarker for de novo lipogenesis (fatty acid synthesis). This method is based upon a stable isotope-dilution technique using LC-MS/MS. MA from 50 μl of serum was derivatized into di-(1-methyl-3-piperidinyl)malonate (DMP-MA) and quantified by LC-MS/MS using the positive electrospray ionization mode. The detection limit of the DMP-MA was approximately 4.8 fmol (500 fg) (signal-to-noise ratio = 10), which was more than 100 times more sensitive compared with that of MA by LC-MS/MS using the negative electrospray ionization mode. The relative standard deviations between sample preparations and measurements made using the present method were 4.4% and 3.2%, respectively, by one-way ANOVA. Recovery experiments were performed using 50 μl aliquots of normal human serum spiked with 9.6 pmol (1 ng) to 28.8 pmol (3 ng) of MA and were validated by orthogonal regression analysis. The results showed that the estimated amount within a 95% confidence limit was 14.1 ± 1.1 pmol, which was in complete agreement with the observed X¯₀ = 15.0 ± 0.6 pmol, with a mean recovery of 96.0%. This method provides reliable and reproducible results for the quantification of MA in human serum.     

LC-ESI-MS determination and pharmacokinetics of adrafinil in rats

A highly sensitive and specific liquid chromatography/tandem mass spectrometric (LC-MS/MS) method for investigating the pharmacokinetics of adrafinil in rats was developed. Rat serum pretreated by solid-phase extraction (SPE) was analyzed by LC-MS/MS with an electrospray ionization (ESI) interface. The mobile phase consisted of acetonitrile:water:acetic acid (35:65:0.1, v/v/v) in an isocratic elution mode pumped at 1.0ml/min. The analytical column (250mmx4.6mm i.d.) was packed with Kromasil C(18) material (5.0mum). The standard curve was linear from 16.5 to 5000ng/ml. The assay was specific, accurate (R.S.D.<2.6%), precise and reproducible (within- and between-day precisions R.S.D. <7.0% and <9.0%, respectively). Adrafinil in rat serum was stable over three freeze-thaw cycles at ambient temperature for 6h. The method had a lower limit of quantitation of 16.5ng/ml, which offered high sensitivity for the determination of adrafinil in serum. The method was successfully applied to pharmacokinetic studies of adrafinil after an oral administration to rats.     

Highly sensitive quantification of key regulatory oxysterols in biological samples by LC-ESI-MS/MS

We describe a highly sensitive and specific method for the quantification of key regulatory oxysterols in biological samples. This method is based upon a stable isotope dilution technique by liquid chromatography-tandem mass spectrometry (LC-MS/MS). After alkaline hydrolysis of human serum (5 microl) or rat liver microsomes (1 mg protein), oxysterols were extracted, derivatized into picolinyl esters, and analyzed by LC-MS/MS using the electrospray ionization mode. The detection limits of the picolinyl esters of 4beta-hydroxycholesterol, 7alpha-hydroxycholesterol, 22R-hydroxycholesterol, 24S-hydroxycholesterol, 25-hydroxycholesterol, 27-hydroxycholesterol, and 24S,25-epoxycholesterol were 2-10 fg (5-25 amol) on-column (signal-to-noise ratio = 3). Reproducibilities and recoveries of these oxysterols were validated according to one-way layout and polynomial equation, respectively. The variances between sample preparations and between measurements by this method were calculated to be 1.8% to 12.7% and 2.9% to 11.9%, respectively. The recovery experiments were performed using rat liver microsomes spiked with 0.05 ng to 12 ng of oxysterols, and recoveries of the oxysterols ranged from 86.7% to 107.3%, with a mean recovery of 100.6%. This method provides reproducible and reliable results for the quantification of oxysterols in small amounts of biological samples.     

Simultaneous determination of myristyl nicotinate, nicotinic acid, and nicotinamide in rabbit plasma by liquid chromatography-tandem mass spectrometry using methyl ethyl ketone as a deproteinization solvent

Myristyl nicotinate (Nia-114) is an ester prodrug being developed for delivery of nicotinic acid (NIC) into the skin for prevention of actinic keratosis and its progression to skin cancer. To facilitate dermal studies of Nia-114, a novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) method using methyl ethyl ketone (MEK) as a deproteinization solvent was developed and validated for the simultaneous determination of Nia-114, NIC, and nicotinamide (NAM) in rabbit plasma. NAM is the principal metabolite of NIC, which is also expected to have chemopreventive properties. The analytes were chromatographically separated using a Spherisorb Cyano column under isocratic conditions, and detected by multiple reaction monitoring (MRM) in positive-ion electrospray ionization mode with a run time of 9 min. The method utilized a plasma sample volume of 0.2 ml and isotope-labeled D4 forms of each analyte as internal standards. The method was linear over the concentration range of 2-1000, 8-1000, and 75-1000 ng/ml, for Nia-114, NIC, and NAM, respectively. The intra- and inter-day assay accuracy and precision were within +/-15% for all analytes at low, medium, and high quality control standard levels. The relatively high value for the lower limit of quantitation (LLOQ) of NAM was demonstrated to be due to the high level of endogenous NAM in the rabbit plasma (about 350 ng/ml). Endogenous levels of NIC and NAM in human, dog, rat, and mouse plasma were also determined, and mean values ranged from <2 ng/ml NIC and 38.3 ng/ml NAM in human, to 233 ng/ml NIC and 622 ng/ml NAM in mouse. Nia-114 was generally unstable in rabbit plasma, as evidenced by loss of 44-50% at room temperature by 2 h, and loss of 64-70% upon storage at -20 degrees C for 1 week, whereas it was stable (<7% loss) upon storage at -80 degrees C for 1 month.     

Liquid chromatography-tandem mass spectroscopy assay for quercetin and conjugated quercetin metabolites in human plasma and urine

A sensitive and specific method was developed and validated for the quantitation of quercetin in human plasma and urine. The application of liquid chromatography-tandem mass spectrometry (LC/MS/MS) with a TurboIonspray (TIS) interface in negative mode under multiple reactions monitoring was investigated. Chromatographic separation was achieved on a C12 column using a mobile phase of acetonitrile/water with 0.2% formic acid (pH 2.4) (40/60, v/v). The detection limit was 100 pg/ml and the lower limit of quantification was 500 pg/ml for plasma samples; the detection limit was 500 pg/ml and the lower limit of quantification was 1 ng/ml for urine samples. The calibration curve was linear from 1 to 800 ng/ml for plasma samples and was linear from 1 to 200 and 50 to 2000 ng/ml for urine samples. All the intra- and inter-day coefficients of variation were less than 11% and intra- and inter-day accuracies were within +/-15% of the known concentrations. This represents a LC/MS/MS assay with the sensitivity and specificity necessary to determine quercetin in human plasma and urine. This assay was used to determine both parent quercetin and the quercetin after enzymatic hydrolysis with beta-glucuronidase/sulfatase in human plasma and urine samples following the ingestion of quercetin 500 mg capsules.     

Simultaneous determination of amitraz and its metabolite in human serum by monolithic silica spin column extraction and liquid chromatography-mass spectrometry

A simple, rapid, sensitive, and specific liquid chromatography-mass spectrometry (LC-MS) method was developed and validated for the quantification of amitraz and its metabolite in human serum. Both the compounds were extracted using monolithic silica spin columns with acetonitrile. The chromatographic separation was performed on a reverse-phase C(18) column with a mobile phase of 10 mM ammonium formate-acetonitrile. The protonated analyte was quantitated in positive ionization by mass spectrometry. The method was validated over the concentration range of 25-1000 ng/ml for amitraz and its metabolite in human serum. For both compounds, the limit of detection was 5 ng/ml. The method was applied to serum samples taken from an attempted suicide patient, and only small volumes of serum were required for the simultaneous determination of these compounds.     

Determination of di(2-ethylhexyl)phthalate and mono(2-ethylhexyl)phthalate in human serum using liquid chromatography-tandem mass spectrometry

Concentrations of mono(2-ethylhexyl)phthalate (MEHP), and di(2-ethylhexyl)phthalate (DEHP), in serum of healthy volunteers were determined by high performance liquid chromatography (HPLC) with tandem mass spectrometry (LC/MS/MS). The serum was extracted with acetone, followed by hexane extraction under acidic conditions, and then applied to the LC/MS/MS. Recoveries of 20 ng/ml of MEHP and DEHP were 101+/-5.7 (n=6) and 102+/-6.5% (n=6), respectively. The limits of quantification (LOQ) of MEHP and DEHP in the method were 5.0 and 14.0 ng/ml, respectively. The concentration of MEHP in the serum was at or less than the LOQ. The concentration of DEHP in the serum was less than the LOQ. Contaminations of MEHP and DEHP from experimental reagents, apparatus and air during the procedure were less than the LOQ and were estimated to be <1.0 and 2.2+/-0.6 ng/ml, respectively. After subtraction of the contamination, the net concentrations of MEHP and DEHP in the serum were estimated at or <5 and <2 ng/ml, respectively. To decrease contamination by DEHP, the cleanup steps and the apparatus and solvent usage were minimized in the sample preparation procedures. The high selectivity of LC/MS/MS is the key for obtaining reliable experimental data from in the matrix-rich analytical samples and for maintaining a low level contamination of MEHP and DEHP in this experimental system. This method would be a useful tool for the detection of MEHP and DEHP in serum.     

Determination of cysteinyl leukotrienes in exhaled breath condensate: method combining immunoseparation with LC-ESI-MS/MS

A rapid and precise method for the identification and quantification of cysteinyl leukotrienes (leukotriene C(4), leukotriene D(4) and leukotriene E(4)), essential markers of bronchial asthma, in exhaled breath condensate was developed. The protocol consists of immunoaffinity separation and a detection step, liquid chromatography combined with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). In particular, the selected reaction monitoring mode was used for its extremely high degree of selectivity and the stable-isotope-dilution assay for its high precision of quantification. The developed method was characterized with a high precision (≤ 7.7%, determined as RSD), an acceptable accuracy (90.4-93.7%, determined as recovery), a low limit of detection (≤ 2 pg/ml EBC) and a low limit of quantification (≤ 10 pg/ml EBC). It was compared to other simple, clinically appropriate combinations of pre-treatment methods (solid phase extraction and lyophilization) with LC/MS. Finally, the method (a combination of immunoaffinity separation with LC-MS) was successfully tested in a clinical study where a significant difference was found in the concentration levels of cysteinyl leukotrienes between patients with occupational bronchial asthma and healthy subjects.     

A rapid, sensitive and selective liquid chromatography/atmospheric pressure chemical ionization tandem mass spectrometry method for determination of fenretinide (4-HPR) in plasma

A simple and sensitive liquid chromatography/tandem mass spectrometry (LC-MS/MS) method using an atmospheric pressure chemical ionization source (APCI) for the quantification of fenretinide (4-HPR) in mouse plasma was developed and validated. After a simple protein precipitation of plasma sample by acetonitrile, 4-HPR was analyzed by LC-APCI-MS/MS. High-performance liquid chromatography (HPLC) separation was conducted on a Hypurity C18 column (50mmx2.1mm, 5microm) with a flow rate 0.60mL/min using a gradient mobile phase comprised of 0.05% formic acid in water (A) and methanol (B), and a run time of 4.5min. The elimination of a tedious sample preparation process and a shorter run time substantially reduced total analysis time. The method was linear over the range 0.5-100ng/mL, with r>0.998. The intra- and inter-assay precisions were 1.4-9.2% and 5.1-8.2%, respectively, and the intra- and inter-assay accuracies were 93.9-98.6% and 92.7-95.3%, respectively. The absolute recoveries were 90.3% (1.5ng/mL), 97.0% (7.5ng/mL) and 92.1% (75.0ng/mL) for 4-HPR, and 99.1% for the internal standard (150ng/mL). The analytical method had excellent sensitivity using a small sample volume (30microL) with the lower limit of quantification (LLOQ) 0.5ng/mL. This method is robust and has been successfully employed in a pharmacokinetic study of 4-HPR in a mouse xenograft model of neuroblastoma.