Determination of ritodrine in human plasma by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry

A simple and sensitive HPLC/MS/MS method was developed and evaluated to determine the concentration of ritodrine (RTD) in human plasma. Liquid-liquid extraction with ethyl acetate was employed as the sample preparation method. The structural analogue salbutamol was selected as the internal standard (IS). The liquid chromatography was performed on a Hanbon Sci. & Tech. Lichrospher CN (150 mm x 4.6 mm, i.d., 5 microm) column (Hanbon, China) at 20 degrees C. A mixture of 0.03% acetic acid and methanol (50:50, v/v) was used as isocratic mobile phase to give the retention time 3.60 min for ritodrine and 2.94 min for salbutamol. Selected reaction monitoring (SRM) in positive ionization mode was employed for mass detection. The calibration functions were linear over the concentration range 0.39-100 ng mL(-1). The intra- and inter-day precision of the method were less than 15%. The lower limit of quantification was 0.39 ng mL(-1). The method had been found to be suitable for application to a pharmacokinetic study after oral administration of 20mg ritodrine hydrochloride tablet to 18 healthy female volunteers. The half-life is 2.54+/-0.67 h.     

Determination of procarbazine in human plasma by liquid chromatography with electrospray ionization mass spectrometry

Procarbazine is a cytotoxic chemotherapeutic agent used in the treatment of lymphomas and brain tumors. Its pharmacokinetic behavior remains poorly understood even though more than 30 years have elapsed since the drug was approved for clinical use. To characterize the pharmacokinetics of procarbazine in brain cancer patients during a phase I trial, a method for determining the drug in human plasma by reversed-phase high-performance liquid chromatography (HPLC) with electrospray ionization mass spectrometry (ESI-MS) was developed and thoroughly validated. Plasma samples were prepared for analysis by precipitating proteins with trichloroacetic acid and washing the protein-free supernatant with methyl tert-butyl ether to remove excess acid. The solution was separated on a Luna C-18 analytical column using methanol-25 mM ammonium acetate buffer, pH 5.1 (22:78, v/v) as the mobile phase at 1.0 ml/min. A single-quadrupole mass spectrometer with an electrospray interface was operated in the selected-ion monitoring mode to detect the [M+H](+) ions at m/z 222.2 for procarbazine and at m/z 192.1 for the internal standard (3-dimethylamino-2-methylpropiophenone). Procarbazine and the internal standard eluted as sharp, symmetrical peaks with retention times (mean+/-S.D.) of 6.3+/-0.1 and 9.9+/-0.3 min, respectively. Calibration curves of procarbazine hydrochloride in human plasma at concentrations ranging from 0.5 to 50 ng/ml exhibited excellent linearity. The mean absolute recovery of the drug from plasma was 102.9+/-1.0%. Using a sample volume of 150 microl, procarbazine was determined at the 0.5 ng/ml (1.9 nM) lower limit of quantitation with a mean accuracy of 105.2% and an interday precision of 3.60% R.S.D. on 11 different days over 5 weeks. During this same time interval, the between-day accuracy for determining quality control solutions of the drug in plasma at concentrations of 2.0, 15 and 40 ng/ml ranged from 97.5 to 98.2% (mean+/-S.D., 97.9+/-0.4%) and the precision was 3.8-6.2% (mean+/-S.D., 5.1+/-1.2%). Stability characteristics of the drug were thoroughly evaluated to establish appropriate conditions to process, store and prepare clinical specimens for chromatographic analysis without inducing significant chemical degradation. The sensitivity achieved with this assay permitted the plasma concentration-time profile of the parent drug to be accurately defined following oral administration of standard doses to brain cancer patients.     

Determination of endocannabinoids in nematodes and human brain tissue by liquid chromatography electrospray ionization tandem mass spectrometry

A simple and highly sensitive liquid chromatography/tandem mass spectrometric (LC/MS/MS) method was developed to compare endogenous cannabinoid levels in nematodes and in brains of rats and humans, with and without prior exposure to ethanol. After liquid-liquid extraction of the lipid fraction from homogenized samples, a reversed-phase sub 2 μm column was used for separating analytes with an isocratic mobile phase. Deuterated internal standards were used in the analysis, and detection was made by triple quadrupole mass spectrometer with multiple reaction monitoring (MRM). Ionization was performed with positive electrospray ionization (ESI). The nematode Caenorhabditis elegans fat-3 mutant, that lacks the necessary enzyme to produce arachidonic acid, the biologic precursor to 2-arachidonoyl glycerol and anandamide, was used as an analyte-free surrogate material for selectivity and calibration studies. The matrix effect was further investigated by in-source multiple reaction monitoring (IS-MRM) and standard addition studies. Selectivity studies demonstrated that the method was free from matrix effects. Good accuracy and precision were obtained for concentrations within the calibration range of 0.4-70 nM and 40-11,000 nM for monitored N-acylethanolamides (NAEs) and acyl glycerols, respectively.     

Determination of soyasaponins Ba and Bb in human serum by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry

A rapid, sensitive and selective high-performance liquid chromatography-tandem mass spectrometric method (HPLC-MS-MS) has been developed and validated for the determination of soyasaponins Ba and Bb in human serum using glycyrrhizin as internal standard (I.S.). Soyasaponins Ba and Bb were extracted from human serum by liquid-liquid extraction and cleaned up by C(18) solid-phase extraction (SPE), followed by separation on a C(18) reversed-phase column using acetonitrile/water containing 0.025% acetic acid as a mobile phase for gradient elution. Soyasaponins Ba and Bb, and I.S. were ionized by negative ion pneumatically assisted electrospray and detected by HPLC-MS-MS in the multiple-reaction monitoring (MRM) mode using precursor-->product ion combinations at m/z 958-->940, 942-->924 and 822-->351, respectively. The calibration curves were linear (r(2)>0.991) in the concentration range of 0.5-100.0 ng/mL, with lower limits of quantification of 0.5 and 0.3 ng/mL for soyasaponins Ba and Bb, respectively, in human serum. Intra-day and inter-day relative standard deviations (R.S.D.) were less than 7.9 and 11.3%, respectively. The mean recoveries of soyasaponins Ba and Bb ranged from 92 to 101% and from 85 to 94%, respectively.     

Analysis of phospholipid species in human blood using normal-phase liquid chromatography coupled with electrospray ionization ion-trap tandem mass spectrometry

A narrow-bore normal-phase high-performance liquid chromatography (HPLC) method was developed for separation of phospholipid classes in human blood. The separation was obtained using an HPLC diol column and a gradient of chloroform and methanol with 0.1% formic acid, titrated to pH 5.3 with ammonia and added 0.05% triethylamine. The HPLC system was coupled on-line with an electrospray ionisation ion-trap mass spectrometer. Chromatographic baseline separation was obtained between phosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, lyso-phosphatidylcholine, phosphatidylinositol and phosphatidylserine, eluting in that order. The total run time was 30 min. Plasmalogen phosphatidylethanolamine and sphingomyelin, which both are substances with structural similarities to the glycerophospholipids, had similar retention time as phosphatidylethanolamine, but were well separated from the other glycerophospholipid classes. The species from each class were identified using MS² or MS³, which forms characteristic lyso-fragments. The combination of lyso-fragment mass, molecular ion and chromatographic retention time was used to identify each species, including 20 species of phosphatidylglycerol. The mass spectra obtained for the phospholipid classes are presented. Using this system 17 disaturated phospholipid species not earlier described to be present in blood were identified. The limit of detection varied between different phospholipid classes and was in the range 0.1–5 ng of injected substance.     

Site-specific N-glycosylation analysis of human plasma ceruloplasmin using liquid chromatography with electrospray ionization tandem mass spectrometry

Ceruloplasmin has ferroxidase activity and plays an essential role in iron metabolism. In this study, a site-specific glycosylation analysis of human ceruloplasmin (CP) was carried out using reversed-phase high-performance liquid chromatography with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). A tryptic digest of carboxymethylated CP was subjected to LC-ESI-MS/MS. Product ion spectra acquired data-dependently were used for both distinction of the glycopeptides from the peptides using the carbohydrate B-ions, such as m/z 204 (HexNAc) and m/z 366 (HexHexNAc), and identification of the peptide moiety of the glycopeptide based on the presence of the b- and y-series ions derived from the peptide. Oligosaccharide composition was deduced from the molecular weight calculated from the observed mass of the glycopeptide and theoretical mass of the peptide. Of the seven potential N-glycosylation sites, four (Asn119, Asn339, Asn378, and Asn743) were occupied by a sialylated biantennary or triantennary oligosaccharide with fucose residues (0, 1, or 2). A small amount of sialylated tetraantennary oligosaccharide was detected. Exoglycosidase digestion suggested that fucose residues were linked to reducing end GlcNAc in biantennary oligosaccharides and to reducing end and/or alpha1-3 to outer arms GlcNAc in triantennary oligosaccharides and that roughly one of the antennas in triantennary oligosaccharides was alpha2-3 sialylated and occasionally alpha1-3 fucosylated at GlcNAc.     

Quantitative determination of mitiglinide in human plasma by ultra-performance liquid chromatography/electrospray ionization tandem mass spectrometry

A selective, rapid and sensitive ultra-performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) method was developed for the quantitative determination of mitiglinide in human plasma. With nateglinide as internal standard, sample pretreatment involved a one-step extraction with diethyl ether of 0.2 mL plasma. The separation was performed on an ACQUITY UPLCtrade mark BEH C(18) column (50 mm x 2.1 mm, i.d., 1.7 microm) with the mobile phase consisting of methanol and 10 mmol/L ammonium acetate (65:35, v/v) at a flow rate of 0.25 mL/min. The detection was carried out by means of electrospray ionization mass spectrometry in positive ion mode with multiple reaction monitoring (MRM). Linear calibration curves were obtained in the concentration range of 1.080-5400 ng/mL, with a lower limit of quantification of 1.080 ng/mL. The intra- and inter-day precision (RSD) values were below 15% and accuracy (RE) was from -3.5% to 7.3% at all QC levels. The method was fully validated and successfully applied to a clinical pharmacokinetic study of mitiglinide in 10 healthy volunteers following oral administration.     

Determination of higenamine in human plasma and urine using liquid chromatography coupled to positive electrospray ionization tandem mass spectrometry

Higenamine is an active ingredient of Aconite root in Chinese herbal medicine and might be used as a new agent for a pharmaceutical stress test and was approved to undergo clinical pharmacokinetic study. Therefore, there exists a need to establish a sensitive and rapid method for the determination of higenamine in human plasma and urine. This paper described a sensitive and rapid method based on liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) for the determination of higenamine in human plasma and urine. Solid-phase extraction (SPE) was used to isolate the compounds from biological matrices followed by injection of the extracts onto an Atlantis dC18 column with isocratic elution. The mobile phase was 0.05% formic acid in water-methanol (40:60, v/v). The mass spectrometry was carried out using positive electrospray ionization (ESI) and data acquisition was carried out in the multiple reaction monitoring (MRM) mode. The method was fully validated over the concentration range of 0.100-50.0 ng/mL and 1.00-500 ng/mL in plasma and urine, respectively. The lower limits of quantification (LLOQs) were 0.100 and 1.00 ng/mL in plasma and urine, respectively. Inter- and intra-batch precision was less than 15% and the accuracy was within 85-115% for both plasma and urine. Extraction recovery was 82.1% and 56.6% in plasma and urine, respectively. Selectivity, matrix effects and stability were also validated in human plasma and urine. The method was applied to the pharmacokinetic study of higenamine hydrochloride in Chinese healthy subjects.     

Quantification of ceramide species in biological samples by liquid chromatography electrospray ionization tandem mass spectrometry

We present an optimized and validated liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) method for the simultaneous measurement of concentrations of different ceramide species in biological samples. The method of analysis of tissue samples is based on Bligh and Dyer extraction, reverse-phase high-performance liquid chromatography separation, and multiple reaction monitoring of ceramides. Preparation of plasma samples also requires isolation of sphingolipids by silica gel column chromatography prior to LC-ESI-MS/MS analysis. The limits of quantification were in a range of 0.01-0.50 ng/ml for distinct ceramides. The method was reliable for inter- and intraassay precision, accuracy, and linearity. Recoveries of ceramide subspecies from human plasma, rat liver, and muscle tissue were 78 to 91%, 70 to 99%, and 71 to 95%, respectively. The separation and quantification of several endogenous long-chain and very-long-chain ceramides using two nonphysiological odd chain ceramide (C17 and C25) internal standards was achieved within a single 21-min chromatographic run. The technique was applied to quantify distinct ceramide species in different rat tissues (muscle, liver, and heart) and in human plasma. Using this analytical technique, we demonstrated that a clinical exercise training intervention reduces the levels of ceramides in plasma of obese adults. This technique could be extended for quantification of other ceramides and sphingolipids with no significant modification.     

Determination of Astragaloside IV in rat plasma by liquid chromatography electrospray ionization mass spectrometry

Astragaloside IV (AGS-IV) is an active constituent of Radix Astragali used in many Traditional Chinese Medicines. This paper describes a sensitive and specific assay for the quantitation of AGS-IV in rat plasma. After solid phase extraction (SPE), samples were analyzed by liquid chromatography electrospray ionization mass spectrometry using a reversed-phase C18 column. The assay was linear in the range 1-500 ng/ml with a limit of detection of 0.5 ng/ml. The recovery was 92.5% and within-day and between-day precision were 3.7-6.0 and 2.8-9.8%, respectively. The assay was applied to a pharmacokinetic study in rat after a single oral dose. The drug was rapidly absorbed and subsequently eliminated according to a biphasic concentration-time curve.     

Determination of vandetanib in human plasma and cerebrospinal fluid by liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS)

A sensitive and precise LC-ESI-MS/MS method for the determination of vandetanib (ZD6474) in human plasma and cerebrospinal fluid (CSF) using [(13)C,d(3)]-ZD6474 as an internal standard (ISTD) was developed and validated. Sample preparation consisted of a simple liquid-liquid extraction with tert-butyl methyl ether containing 0.1% or 0.5% ammonium hydroxide. ZD6474 and ISTD were separated on a Kinetex C18 column (2.6 μm, 50 mm × 2.1 mm) at ambient temperature with an isocratic mobile phase (acetonitrile/10mM ammonium formate=50/50, v/v, at pH 5.0) delivered at 0.11 mL/min. The retention time of both compounds was at 1.60 min in a runtime of three min. Detection was achieved by an API-3200 LC-MS/MS system, monitoring m/z 475.1/112.1 and m/z 479.1/116.2 for vandetanib and ISTD, respectively. The method was linear in the range of 0.25-50 ng/mL (R(2) ≥ 0.990) for the CSF curve and from 1.0 to 3000 ng/mL (R(2) ≥ 0.992) for the plasma curve. The mean recovery for vandetanib was 80%. Within-day and between-day precisions were ≤ 8.8% and ≤ 5.9% for CSF and plasma, respectively. Within-day and between-day accuracies ranged from 95.0 to 98.5% for CSF, and from 104.0 to 108.5% for plasma. Analysis of plasma from six different sources showed no matrix effect for vandetanib (MF=0.98, %CV ≤ 4.97, n=6). This method was successfully applied to the analysis of pharmacokinetic samples from children with brain tumors treated with oral vandetanib.     

Quantitative statistical analysis of standard and human blood proteins from liquid chromatography, electrospray ionization, and tandem mass spectrometry

It will be important to determine if the parent and fragment ion intensity results of liquid chromatography, electrospray ionization and tandem mass spectrometry (LC-ESI-MS/MS) experiments have been randomly and independently sampled from a normal population for the purpose of statistical analysis by general linear models and ANOVA. The tryptic parent peptide and fragment ion m/z and intensity data in the mascot generic files from LC-ESI-MS/MS of purified standard proteins, and human blood protein fractionated by partition chromatography, were parsed into a Structured Query Language (SQL) database and were matched with protein and peptide sequences provided by the X!TANDEM algorithm. The many parent and/or fragment ion intensity values were log transformed, tested for normality, and analyzed using the generic Statistical Analysis System (SAS). Transformation of both parent and fragment intensity values by logarithmic functions yielded intensity distributions that closely approximate the log-normal distribution. ANOVA models of the transformed parent and fragment intensity values showed significant effects of treatments, proteins, and peptides, as well as parent versus fragment ion types, with a low probability of false positive results. Transformed parent and fragment intensity values were compared over all sample treatments, proteins or peptides by the Tukey-Kramer Honestly Significant Difference (HSD) test. The approach provided a complete and quantitative statistical analysis of LC-ESI-MS/MS data from human blood.     

Simultaneous measurement of tryptophan and related compounds by liquid chromatography/electrospray ionization tandem mass spectrometry

We have expanded a liquid chromatographic-tandem mass spectrometric method that measures 3-hydroxykynurenine and 3-hydroxyanthranilic acid in addition to tryptophan and kynurenine both intra- and extracellularly. After reversed phase HPLC separation, the compounds were detected in the MS positive multiple reaction monitoring mode. We found a good linear response for each tryptophan metabolite. The lower limit of quantification for each compound ranged from 0.01 to 0.1 microM. The extraction efficiencies from spiked cell samples and culture medium ranged between 83 and 111% and the overall coefficient of variation of analyses was less than 7%. Using our method, we found tryptophan metabolites in the cells and the culture medium of LN229 human glioma cells were stimulated by interferon-gamma, a known inducer of indoleamine 2,3-dioxygenase. The intracellular concentrations of kynurenine, 3-hydroxykynurenine and 3-hydroxyanthranilic acid were higher than those in the medium. This is the first report of a method for the simultaneous determination of tryptophan and its metabolic products both intra- and extracellularly.     

Determination of glimepiride in human plasma by liquid chromatography-electrospray ionization tandem mass spectrometry

A sensitive and specific high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS-MS) method has been developed at our center for the determination of glimepiride in human plasma. After the addition of the internal standard, plasma samples were extracted by liquid-liquid extraction technique using diethyl ether. The compounds were separated on a prepacked C18 column using a mixture of acetonitrile, methanol and ammonium acetate buffer as mobile phase. A Finnigan LCQDUO ion trap mass spectrometer connected to an Alliance Waters HPLC was used to develop and validate the method. The analytical method was validated according to the FDA bioanalytical method validation guidance. The results were within the accepted criteria as stated in the aforementioned guidance. The method was proved to be sensitive and specific by testing six different plasma batches. Linearity was established for the range of concentrations 5.0-500.0 ng/ml with a coefficient of determination (r2) of 0.9998. Accuracy for glimepiride ranged from 100.58 to 104.48% at low, mid and high levels. The intra-day precision was better than 12.24%. The lower limit of quantitation (LLOQ) was identifiable and reproducible at 5.0 ng/ml with a precision of 7.96%. The proposed method enables the unambiguous identification and quantitation of glimepiride for pharmacokinetic, bioavailability or bioequivalence studies.     

High-throughput quantification of soy isoflavones in human and rodent blood using liquid chromatography with electrospray mass spectrometry and tandem mass spectrometry detection

Soy-containing foods and dietary supplements are widely consumed for putative health benefits (e.g., cancer chemoprevention, beneficial effects on serum lipids associated with cardiovascular health, reduction of osteoporosis, relief of menopausal symptoms). However, studies of soy isoflavones in experimental animals suggest possible adverse effects as well (e.g., enhancement of reproductive organ cancer, modulation of endocrine function, anti-thyroid effects). This paper describes the development and validation of a sensitive high throughput method for quantifying isoflavones in blood from experimental animal and human studies. Serum samples containing genistein, daidzein, and equol were processed using reverse phase solid-phase extraction in the 96-well format for subsequent LC-ES/MS/MS or LC-ES/MS analysis using isotope dilution in conjunction with labeled internal standards. The method was validated by repetitive analysis of spiked blank serum and the intra-day and inter-day accuracy (88-99%) and precision (relative standard deviations from 3 to 13%) of measurement determined. The lower limit of quantification for all isoflavones was approximately 0.005 micro M using MS/MS detection, and 0.03 micro M using MS for genistein and daidzein. The degree of method performance, with respect to throughput, sensitivity and selectivity, makes this approach practical for analysis of large sample sets generated from mechanistic animal studies and human clinical trials of soy isoflavones.     

Quantification of roxatidine in human plasma by liquid chromatography electrospray ionization tandem mass spectrometry: application to a bioequivalence study

A sensitive and specific method using a one-step liquid-liquid extraction (LLE) with ethyl acetate followed by high-performance liquid chromatography (HPLC) coupled with positive ion electrospray ionization tandem mass spectrometry (ESI-MS/MS) detection was developed and validated for the determination of roxatidine in human plasma using famotidine as an internal standard (IS). Data acquisition was carried out in multiple reaction monitoring (MRM) mode, by monitoring the transitions m/z 307.3-->107.1 for roxatidine and m/z 338.4-->189.1 for famotidine. Chromatographic separation was performed on a reverse phase Hydrosphere C(18) column at 0.2 mL min(-1) using a mixture of methanol-ammonium formate buffer as mobile phase (20:80, v/v; adjusted to pH 3.9 with formic acid). The achieved lower limit of quantification (LLOQ) was 1.0 ng mL(-1) and the standard calibration curve for roxatidine was linear (r(2)=0.998) over the studied range (1-1000 ng mL(-1)) with acceptable accuracy and precision. Roxatidine was found to be stable in human plasma samples under short-, long-term storage and processing conditions. The developed method was validated and successfully applied to the bioequivalence study of roxatidine administrated as a single oral dose (75 mg as roxatidine acetate hydrochloride) to healthy female Korean volunteers.     

Determination of olopatadine, a new antiallergic agent, and its metabolites in human plasma by high-performance liquid chromatography with electrospray ionization tandem mass spectrometry

A rapid, sensitive and specific assay method has been developed to determine plasma concentrations of olopatadine hydrochloride (A) and its metabolites, M1 (B), M2 (C) and M3 (D), using high-performance liquid chromatography with electrospray ionization tandem mass spectrometry (LC–ESI-MS–MS). Olopatadine, its metabolites, and internal standard, KF11796 (E), were separated from plasma using solid-phase extraction (Bond Elut C₁₈ cartridge). The eluate was dried, reconstituted and injected into the LC–ESI-MS–MS system. The calibration curves showed good linearity over the ranges 1–200 ng/ml for olopatadine and M3, and 2–100 ng/ml for M1 and M2, and the method was thoroughly validated and applied to the determination of olopatadine and its metabolites in plasma collected during Phase I clinical trials. Furthermore, the assay values were compared with those determined by the radioimmunoassay method, which has been routinely used to determine olopatadine in plasma.     

Quantitative determination of the diastereoisomers of hexabromocyclododecane in human plasma using liquid chromatography coupled with electrospray ionization tandem mass spectrometry

A sensitive, simple and feasible method has been developed and validated for the simultaneous determination of three diastereoisomers of hexabromocyclododecane (HBCD) in human plasma using liquid chromatography tandem mass spectrometry (LC-MS/MS). The simple pretreatment generally involved protein precipitation with methanol (MeOH). The separation was performed with a C18 reverse phase column. The mobile phases were 5mM ammonium acetate (NH(4)AC) in water and acetonitrile (ACN). The mass spectrometer was operated using negative electrospray ionization (ESI) source and the data acquisition was carried out with multiple reaction monitoring (MRM) mode. The analyte quantifications were performed by external standard method with matrix-matched calibration curves. The method was partially validated with the evaluations of accuracy, precision, linearity, limit of quantification (LOQ), limit of detection (LOD), recovery, matrix effect and carryover effect. With the present method, the intra-batch accuracies were 94.7-104.3%, 91.9-109.3% and 89.8-105.0% for α-, β- and γ-HBCD, respectively. And the inter-batch accuracies were ranged from 94.2% to 109.7%. Both intra-batch and inter-batch precisions (relative standard deviation, RSD, %) of the analytes were no more than 11.2%. The recoveries were from 79.0% to 108.9% and the LOQ was 10pg/mL for each diastereoisomer. The linear range was 10-10,000pg/mL with the linear correlation coefficient R(2)>0.996. No significant matrix effect and carryover effect of the analytes were observed in this study. This method is in possession of sufficient resolution, high sensitivity as well as selectivity and convenient to be applied to the trace determination of HBCDs in human plasma.     

Determination of iodide in urine using electrospray ionization tandem mass spectrometry

A rapid and sensitive electrospray ionization (ESI) tandem mass spectrometry (MS–MS) procedure was developed for the determination of iodide (I⁻). A gold (Au) and I⁻ complex was formed immediately after the addition of the chelating agent NaAuCl₄ to I⁻ solution, and was extracted with methyl isobutyl ketone. One to five microliters of the extract were injected directly into an ESI–MS–MS instrument. I⁻ quantification was performed by selecting reaction monitoring of the product ion I⁻ at m/z 127 derived from the precursor ion ¹⁹⁷AuI₂⁻ at m/z 451. I⁻ concentration was measured in the quantification range from 10⁻⁷ to 10⁻⁵ M using 50 μL of solution within 10 min. Iodate was reduced to I⁻ with ascorbic acid and determined. I⁻ concentration in reference urine 2670a was measured after treatments.     

Determination of azatadine in human plasma by liquid chromatography/tandem mass spectrometry

A sensitive method using liquid chromatography with tandem mass spectrometric detection (LC-MS/MS) was developed and validated for the analysis of antihistamine drug azatadine in human plasma. Loratadine was used as internal standard (IS). Analytes were extracted from human plasma by liquid/liquid extraction using ethyl acetate. The organic phase was reduced to dryness under a stream of nitrogen at 30 °C and the residue was reconstituted with the mobile phase. 5 μL of the resulting solution was injected onto the LC-MS/MS system. A 4.6 mm × 150 mm, I.D. 5 μm, Agilent TC-C(18) column was used to perform the chromatographic analysis. The mobile phase consisted of ammonium formate buffer 0.010 M (adjusted to pH 4.3 with 1M formic acid)/acetonitrile (20:80, v/v) The chromatographic run time was 5 min per injection and flow rate was 0.6 mL/min. The retention time was 2.4 and 4.4 min for azatadine and IS, respectively. The tandem mass spectrometric detection mode was achieved with electrospray ionization (ESI) iron source and the multiple reaction monitoring (MRM) (291.3 → 248.2m/z for azatadine, 383.3 → 337.3m/z for IS) was operated in positive ion modes. The low limit of quantitation (LLOQ) was 0.05 ng/mL. The intra-day and inter-day precision of the quality control (QC) samples was 8.93-11.57% relative standard deviation (RSD). The inter-day accuracy of the QC samples was 96.83-105.07% of the nominal values.