Rapid and Sensitive LC–MS/MS Analysis of Fatty Acids in Clinical Samples

Free fatty acids (FFAs), major cellular metabolites, play an important role during tumor pathogenesis. Enhanced de novo fatty acid synthesis in tissues is a characteristic feature of cancer. Therefore, measurement of FFA concentration in biological samples is beneficial for cancer research and clinical diagnosis. Herein, a rapid, stable, and sensitive detection methodology was established to simultaneously quantify 22 FFAs using high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC/ESI–MS/MS). The HPLC–MS/MS system was run in negative ion mode for 15 min using multiple reaction monitoring. The lipids were extracted from colon tissues of colon cancer patients and then injected into the HPLC–MS/MS system for analysis. Colon samples were analyzed by inter-day repeatability and intra-day repeatability, with less than 5 % deviation for most fatty acids. This approach is successful to determine low picogram concentrations of each FFA molecule using milligrams of tissue, and provides a promising method for FFA microanalysis in clinical samples.     

LC/MS and LC/MS/MS determination of protein tryptic digests

Tryptic digests were analyzed by means of online microbore liquid chromatography combined with mass spectrometry (LC/MS) for some common proteins. Following conventional enzymatic digestion with trypsin, the freeze-dried residues were dissolved in high-performance liquid chromatography (HPLC) eluent and subjected to gradient reversed-phase microbore HPLC separation with mass spectrometric detection. The latter was done in the full-scan single or tandem (MS/MS) mass spectrometry mode. The formation of gas-phase ions from dissolved analytes was accomplished at atmospheric pressure by pneumatically assisted electrospray (ion spray) ionization. This produced field-assisted ion evaporation of dissolved ions, which could then be mass-analyzed for molecular mass or structure. In the full-scan LC/MS mode, the masses for the peptide fragments in the tryptic digests can be determined as either their singly or multiply charged ions. When the molecular weights of the peptides lie outside the mass range of the mass spectrometer, the multiply charged feature of these experimental conditions still provides reliable molecular weight determinations. In addition, collision-activated dissociation (CAD) on selected peptide precursor ions provides online LC/MS/MS sequence information for the tryptic fragments. Results are shown for the tryptic digests of horse heart cytochrome c, bovine β-lactoglobulin A, and bovine β-lactoglobulin B.     

Sequential ortho effects: characterization of novel [M - 35]+ fragment ions in the mass spectra of 2-alkyl-4, 6-dinitrophenols

High-resolution mass spectrometry (HRMS), hybrid tandem mass spectrometry (MS/MS) (EBqQ), and photoelectron-photoion coincidence (PEPICO) experiments were conducted to examine a possible ortho-ortho effect resulting in a novel [M - 35]⁺ fragment ion in 2-alkyl-4, 6-dinitrophenols. For compounds having ethyl or larger alkyl substituents, [M35]⁺ was observed only when [M - 18]⁺ ions were present, with the ortho nitro group being involved in the reaction to [M- 35]⁺. For [M - 18]⁺ and [M - 35]⁺, HRMS results were consistent with losses of H₂O and H₂O + OH, respectively, whereas MS/MS results indicated a sequential reaction due to metastable dissociations. The appearance energy determined by PEPICO for [M - 35]⁺ was found to be greater than the appearance energy for [M - 18]⁺, thus supporting a sequential reaction. 69–75).     

Analytical approaches for the determination of PCB metabolites in blood: a review

Polychlorinated biphenyls (PCBs) are among the most ubiquitous pollutants in the environment, and their metabolism leads to the formation of hydroxylated PCBs (OH-PCBs) and methyl sulfone PCBs (MeSO₂-PCBs). These metabolites are generally more hydrophilic than the parent compound, and therefore are more easily eliminated from the body. However, some congeners have been shown to be strongly retained in human blood, binding to transthyretin with an affinity that is, in general, greater than that of the natural ligand thyroxin itself, which could result in toxicological effects, particularly on the thyroid system. Currently available analytical methods require, in general, extensive sample preparation, which includes a series of time-consuming and low-throughput liquid–liquid and back extractions, evaporations, several cleanup steps, and in some cases, derivatization prior to analysis by gas chromatography (GC) or liquid chromatography (LC) coupled with mass spectrometry (MS). Recent developments in the use of LC coupled with tandem MS (MS/MS) have brought some improvements in terms of sample preparation for the determination of PCB metabolites in blood, although there are still possibilities for continued development. The selected literature has evidenced few studies of LC–MS/MS-based methods, a lack of analytical standards, nonassessment of lower-chlorinated OH-PCBs, and scarce attention to MeSO₂-PCBs in blood. This review aims to evaluate critically the currently available analytical methods for determination of OH-PCBs and MeSO₂-PCBs in blood.     

Stereoselective separation and pharmacokinetic dissipation of the chiral neonicotinoid sulfoxaflor in soil by ultraperformance convergence chromatography/tandem mass spectrometry

Ultraperformance convergence chromatography/tandem triple quadrupole mass spectrometry (UPC²-MS/MS) is a novel tool in separation science that combines the advantages of supercritical fluid chromatography with ultraperformance liquid chromatography/MS/MS technology. The use of nontoxic CO₂ fluid and a postcolumn additive to complement MS/MS allows better control of analyte retention for chiral separation and high-sensitivity determination with different chiral stationary phases. This paper reports the stereoselective separation and determination of the chiral neonicotinoid sulfoxaflor in vegetables and soil by UPC²-MS/MS. Baseline resolution (Rs?≥?1.56) of and high selectivity (LOQ?≤?1.83 μg/kg) for the four stereoisomers were achieved by postcolumn addition of 1 % formic acid–methanol to a Chiralpak IA-3 using CO₂/isopropanol/acetonitrile as the mobile phase at 40 °C, 2,500 psi, and for 6.5 min in electrospray ionization positive mode. Rearranged Van’t Hoff equations afforded the thermodynamic parameters ΔH ^ο and ΔS ^ο, which were analyzed to promote understanding of the enthalpy-driven separation of sulfoxaflor stereoisomers. The interday mean recovery, intraday repeatability, and interday reproducibility varied from 72.9 to 103.7 %, from 1.8 to 9.2 %, and from 3.1 to 9.4 %, respectively. The proposed method was used to study the pharmacokinetic dissipation of sulfoxaflor stereoisomers in soil under greenhouse conditions. The estimated half-life ranged from 5.59 to 6.03 d, and statistically nonsignificant enantioselective degradation was observed. This study not only demonstrates that the UPC²-MS/MS system is an efficient and sensitive method for sulfoxaflor stereoseparation, but also provides the first experimental evidence of the pharmacokinetic dissipation of sulfoxaflor stereoisomers in the environment. Graphical Abstract

Chemical structure and UPC²-MS/MS separation chromatogram of sulfoxaflor. (* stereogenic center)     

Laser diode thermal desorption atmospheric pressure chemical ionization tandem mass spectrometry applied for the ultra-fast quantitative analysis of BKM120 in human plasma

A sensitive and ultra-fast method utilizing the laser diode thermal desorption ion source using atmospheric pressure chemical ionization coupled to tandem mass spectrometry (LDTD-APCI-MS/MS) was developed for the quantitative analysis of BKM120, an investigational anticancer drug in human plasma. Samples originating from protein precipitation (PP) followed by salting-out assisted liquid-liquid extraction (SALLE) were spotted onto the LazWell? plate prior to their thermal desorption and detection by tandem mass spectrometry in positive mode. The validated method described in this paper presents a high absolute extraction recovery (>90 %) for BKM120 and its internal standard (ISTD) [D₈]BKM120, with precision and accuracy meeting the acceptance criteria. Standard curves were linear over the range of 5.00 to 2000 ng mL^?1 with a coefficient of determination (R ²) >0.995. The method specificity was demonstrated in six different batches of human plasma. Intra- and inter-run precision as well as accuracy within ±20 % at the lower limit of quantification (LLOQ) and ±15 % (other levels) were achieved during a three-run validation for quality control (QC) samples. The post-preparative stability on the LazWell? plate at room temperature was 72 h and a 200-fold dilution of spiked samples was demonstrated. The method was applied successfully to three clinical studies (n?=?847) and cross-checked with the validated LC-ESI-MS/MS reference method. The sample analysis run time was 10 s as compared to 4.5 min for the current validated LC-ESI-MS/MS method. The resultant data were in agreement with the results obtained using the validated reference LC-ESI-MS/MS assay and the same pharmacokinetic (PK) parameters were calculated for both analytical assays. This work demonstrates that LDTD-APCI-MS/MS is a reliable method for the ultra-fast quantitative analysis of BKM120 which can be used to speed-up and support its bioanalysis in the frame of the clinical trials.     

Identification and monitoring of thiabendazole transformation products in water during Fenton degradation by LC-QTOF-MS

This work enabled the identification of major transformation products (TPs) of thiabendazole (TBZ) during the Fenton process. TBZ is a benzimidazole fungicide widely used around the world to prevent and/or treat a wide range of fruit and vegetable pathogens. The degradation of the parent molecule and the identification of the main TPs were carried out in demineralized water. The TPs were monitored and identified by liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QTOF-MS/MS). Up to 12 TPs were tentatively identified. Most of them were eliminated after 15 min of treatment time and originated from numerous hydroxylations undergone by the aromatic ring during the initial stages of the process.     

Rapid detection of pesticides not amenable to multi-residue methods by flow injection–tandem mass spectrometry

Flow injection combined with tandem mass spectrometry (MS/MS) was investigated for the rapid detection of highly polar pesticides that are not amenable to multi-residue methods because they do not partition into organic solvents and require dedicated chromatographic conditions. The pesticides included in this study were amitrole, chlormequat, cyromazine, daminozide, diquat, ethephon, fosetyl-Al, glufosinate, glyphosate and its metabolite aminomethylphosphonic acid, maleic hydrazide, mepiquat and paraquat. The composition of the flow-injection solvent was optimized to achieve maximum MS/MS sensitivity. Instrumental limits of detection varied between <0.05 and 1 pg. Fruit, vegetable, cereal, milk and kidney samples were extracted with water (1 % formic acid in case of paraquat/diquat) and ten times diluted in either methanol/0.1 % formic acid, methanol/0.1 % ammonia or acetonitrile/0.1 % ammonia, depending on the pesticide. The ion suppression observed depended strongly on both the matrix and the pesticide. This could be largely compensated for by matrix-matched calibration, but more accurate quantification was obtained by using isotopically labelled standards (commercially available for most of the pesticides studied). The method detection limits ranged from 0.02 mg/kg for chlormequat and mepiquat to 2 mg/kg for maleic hydrazide and were 0.05–0.2 mg/kg for most other pesticide/matrix combinations. This was sufficiently low to test compliance with EU maximum residue limits for many relevant pesticide/commodity combinations. The method substantially reduces the liquid chromatography–MS/MS capacity demand which for many laboratories is prohibitive for inclusion of these pesticides in their monitoring and surveillance programmes. Figure

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Isotope dilution gas chromatography/mass spectrometry for platinum determination in urine

The therapeutic importance of platinum (Pt) compounds, the growing accessibility of gas chromatography/mass spectrometry (GC/MS) systems in clinical laboratories, and the lack of a mass spectrometric method for the determination of Pt in biological samples motivated us to develop an isotope dilution GC/MS assay for Pt. The method is based on the use of lithium bis(trifluoroethyl) dithiocarbamate, Li(FDEDTC), as a chelating agent and enriched ¹⁹²Pt for isotope dilution. Conditions were optimized for the precise and accurate determination of isotope ratios of Pt by using a 10-m DB-l fused silica capillary column and a reverse-geometry double-focusing mass spectrometer with selected ion monitoring. An overall precision of 1% was obtained by combining within-run precision and between-run precision at the 10-ng level. No appreciable memory effect was observed when samples with different isotope ratios were analyzed sequentially. The method was validated by the quantitation of Pt in National Institute of Standards and Technology freeze-dried urine sample SRM 2670. A concentration value of 125 ± 6 /Lg/L (n = 6) was obtained by using four different sets of isotope ratios in the molecular ion and supports the National Institute of Standards and Technology recommended value of 120 ± ? μg/L. Limits-of-quantitation, estimated at 3 μg/L, are made possible by the high sensitivity of the method and the low blank value for Pt.     

Quantification of soy isoflavones and their conjugative metabolites in plasma and urine: an automated and validated UHPLC-MS/MS method for use in large-scale studies

The biotransformation of isoflavones by gut microbiota and by drug metabolizing enzymes plays a crucial role in the understanding of their potential health-promoting effects. The purpose of our work was to develop a simultaneous, sensitive, and robust automated ultra high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method to quantify the soy isoflavones daidzein and genistein, their conjugative metabolites, as well as their major microbial degradation products in order to provide a method for use in large clinical trials or animal studies. An automated, 96-well solid-phase extraction method was used to extract the isoflavone analytes from plasma and urine. Separation of genistein, daidzein, and 19 of its metabolites, including five glucuronides, seven sulfates, and two sulfoglucuronides, as well as five microbial metabolites, was achieved in less than 25 min using a sub-2 μm particle column and a gradient elution with acetonitrile/methanol/water as mobile phases. Analysis was performed under negative ionization electrospray MS via the multiple reaction monitoring (MRM). Validation was performed according to the analytical method validation guidelines of Food and Drug Administration (FDA) and International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) consisting of selectivity, accuracy, precision, linearity, limit of detection, recovery, matrix effect, and robustness. All validated parameters essentially matched the FDA and ICH requirements. The application of this method to a pharmacokinetic study in postmenopausal women showed that isoflavones are extensively metabolized in vivo. A robust automated analytical approach was developed, which allows the handling of large sample sizes but nevertheless provides detailed information on the isoflavone metabolite profile leading to a better understanding and interpretation of clinical and animal studies.     

Combined capillary gas chromatography/ion trap mass spectrometry quantitative methods using labeled or unlabeled internal standards

Three methods of operating an ion trap mass spectrometer (ITMS) were investigated for the determination of quantitative information by combined capillary GC/MS and GC/MS/MS. Separate regression Lines for a hexynone drug were examined by using bath unlabeled and stable-isotope-labeled internal standards. The ITMS modes of operation examined were (1) the full-scan, rf-voltage-only mode, useful on ion trap detectors as well as on the ITMS, (2) a scan using combined rf and dc voltages (rf/dc) for mass-selective storage analyses, and (3) rf/dc followed by collision-induced dissociation, Results of over 200 analyses in the 0.1–10 ng on-column range, with 5 ng of internal standard, showed that the unlabeled internal standard, separated by retention time on the capillary GC column, gave the best relative standard deviatiod (less than 5% over the range) and linear correlations (r ² typically > 0.9992).     

Comparison of Different Sorbents in the QuEChERS Method for the Determination of Pesticide Residues in Strawberries by LC–MS/MS

A comparison of sample preparation based on the quick, easy, cheap, effective, rugged and safe (QuEChERS) method for analysis of pesticide residues in strawberries by LC–MS/MS was made using different sorbents in the clean-up by dispersive solid-phase extraction (d-SPE). Some sorbents were laboratory-made, prepared by depositing poly(methyloctadecylsiloxane) (PMODS), poly(methyloctylsiloxane) (PMOS), aminopropyl-terminated poly(dimethylsiloxane) (APPS) and copolymer of (52–48 %)dimethyl-(48–52 %)methylphenyl-siloxane (DMMPS) onto silica supports. The commercial sorbent primary–secondary amine (PSA) and mixtures of two sorbents, primary–secondary amine and poly(methyloctadecylsiloxane), were also used in the experiments. The performances of the sorbents were evaluated by parameters such as color of the final extract, gravimetric measurement, recovery and matrix effect at the fortification level of 100 ng g^?1 of the pesticide mixture in strawberries. The recoveries were in the range 70–120 %, and the RSD values were lower than 20 % for most of the pesticides using the modified QuEChERS method with different sorbents in the clean-up step. The strawberry extracts were cleaned more efficiently with the use of primary–secondary amine sorbent, which has the function of removing sugars, organic acids and especially pigments. The sample preparation method was efficient, and LC–MS/MS determination was optimal because of high selectivity and good detectivity for the multiresidue analyses.     

Molecular species analysis of arachidonate containing glycerophosphocholines by tandem mass spectrometry

Carboxylate anions arising from collision-induced dissociation (CID) of the [M - 15]^- ion produced by fast atom bombardment (FAB) of glycerophosphocholine (GPCho) were previously shown to be produced in an abundance ratio of 1:3 for the carboxylic acids esterified at sn - 1 and sn - 2, respectively. This observation has been confirmed in a series of 13 synthetic GPCho molecular species. A good correlation was found between the isomeric purity of GPCho molecular species as determined by negative-ion FAB/CID analysis and the isomeric purity of the sn - 2 fatty acid using a phospholipase A₂ assay. Negative-ion FAB mass spectra of several 1-0-alkyl-2-acyl-GPCho molecular species were found to be similar to those of diacyl GPCho. However, the cm spectra from the major high-mass ions are different from those of the diacyl species in that the [M - 15]^- ion yields only one carboxylate anion and the [M - 86]^- undergoes a neutral loss of the sn - 2 carboxylic acid as a major decomposition product. These results suggest several rules useful for structural characterization of GPCho molecular species by negative-ion tandem mass spectrometry (MS/MS): (1) For diacyl species, the mass of the two carboxyl anions plus the mass of the GPeho backbone (minus a methyl group) must correspond to the mass of the [M - 15] anion; (2) for diacyl species there is a carboxylate anion ratio approximately 1:3 for the substituents at sn - 1 and sn - 2; and (3) for alkylether species, only one fatty acyl group is present, and the difference between the [M - 15] ion and the GPCho backbone (minus methyl) plus the fatty acyl group at sn - 2 corresponds to an alkylether substituent. (4) Assignment of ether-linked molecular species can be made from the [M - 86]^- ion, which has a strong neutral loss of the sn - 2 fatty acid. Analysis of GPCho isolated from human neutrophils by total lipid extraction and normal-phase HPLC was carried out by negative-ion FABand MS/MS. The major arachidonate-eontaining molecular species, which comprise only 5% of total GPCho, were identified by using precursor ion scans for the arachidonate anion, m/ z 303. Decomposition of identified. precursor ions permitted the assignment of those molecular species of GPCho that contain arachidonate at sn - 2 and identification of the substituent at the sn - 1 position. These results were compared to previously identified molecular species from human neutrophils. Several minor arachidonate-containing molecular species were tentatively identified.     

In vitro/in vivo screening of oxidative homeostasis and damage to DNA,protein, and lipids using UPLC/MS-MS

Multiple analytical methods are required to comprehensively assess oxidative homeostasis and specific damage to macromolecules. Our aim was to develop a straightforward strategy for the fast assessment of global oxidative status and specific damage to DNA, proteins, and lipids. To this end, an analytical method, based on ultra-performance liquid chromatography coupled to mass spectrometry (UPLC-MS/MS), was developed and validated for the quantification of 16 oxidative stress (OS) biomarkers. Some of these markers were unstable; thus, an easy sample treatment procedure, including fractionation and derivatization, was set up. The method was validated according to Food and Drug Administration (FDA) guidelines, and it provided good results in terms of intra- and inter-day precision (≤17.2 and 16 %, respectively), accuracy (relative error measurement between ?16.6 and 19.8 %), and linearity (R ²?>?0.994). The approach was applied to determine the oxidative insult provoked to cultured rat hepatocytes by cumene hydroperoxide and to analyze the liver and serum samples from patients diagnosed with nonalcoholic steatohepatitis. In both studies, significant differences were found if compared to the corresponding control groups; interestingly, ophthalmic acid was shown as an OS biomarker in both models for the first time. A key advantage of the novel approach in comparison with former multi-method approaches is that now a single method is applied to assess the 16 OS biomarkers. Its comprehensive capacity to profile oxidative homeostasis and damage in both in vitro and clinical samples has been illustrated, which indicates that the proposed approach is a good choice to evaluate whether OS is involved in physiological signals, diseases, or toxic events and to what extent.     

Simultaneous determination of reduced and oxidized glutathione in tissues by a novel liquid chromatography-mass spectrometry method: application in an inhalation study of Cd nanoparticles

The paper presents the development of an advanced extraction and fast analytical LC MS/MS method for simultaneous analyses of reduced and oxidized glutathione (GSH and GSSG, respectively) in different animal tissues. The simultaneous determination of GSH and GSSG is crucial because the amount and ratio of both GSH and GSSG may be altered in response to oxidative stress, an important mechanism of toxicity. The method uses the derivatization of free thiol groups in GSH. Its performance was demonstrated for less explored tissues (lung, brain, and liver) in mouse. The combined extraction and analytical method has very low variability and good reproducibility, maximum coefficients of variance for within-run and between-run analyses under 8 %, and low limits of quantification; for GSH and GSSG, these were 0.2 nM (0.06 ng/mL) and 10 nM (6 ng/mL), respectively. The performance of the method was further demonstrated in a model experiment addressing changes in GSH and GSSG concentrations in lung of mice exposed to CdO nanoparticles during acute 72 h and chronic 13-week exposures. Inhalation exposure led to increased GSH concentrations in lung. GSSG levels were in general not affected; nonsignificant suppression occurred only after the longer 13-week period of exposure. The developed method for the sensitive detection of both GSH and GSSG in very low tissue mass enables these parameters to be studied in cases where only a little sample is available, i.e. in small organisms or in small amounts of tissue.     

Metabolic fingerprinting based on high-resolution tandem mass spectrometry: a reliable tool for wine authentication?

Ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (MS) and an alternative technology represented by direct analysis in real time coupled with quadrupole time-of-flight MS were investigated for metabolic fingerprinting of 343 red and white wine samples. Direct injection of pure wine and an extraction procedure optimized for isolation of polyphenols were used to compare different analytical and data handling strategies. After data processing and data pretreatment, principal component analysis was initially used to explore the data structure. Initially, the unsupervised models revealed a notable clustering according to the grape varieties, and therefore supervised orthogonal partial least squares discriminant analysis models were created and validated for separation of red and white wines according to the grape variety. The validated orthogonal partial least squares discriminant analysis models based on data (ions) recorded in positive ionization mode were able to classify correctly 95 % of samples. In parallel, authentication parameters, such as origin and vintage, were evaluated, and they are discussed. A tentative identification of markers was performed using accurate mass measurement of MS and MS/MS spectra, different software packages and different online libraries. In this way, different flavonol glucosides and polyphenols were identified as wine markers according to the grape varieties.     

Determination of α-chaconine and α-solanine in commercial potato crisps by QuEChERS extraction and UPLC-MS/MS

A simple and fast analytical method for the determination of the main steroidal glycoalkaloids, α-chaconine and α-solanine, in commercial potato crisps, based on QuEChERS (Quick, Easy, Cheap, Effective, Rugged, Safe) extraction and ultra performance liquid chromatography coupled with an electrospray ionization triple-quadrupole tandem mass spectrometer (UPLC-MS/MS) in the multiple reaction monitoring mode was established and validated. The sample preparation procedure involves the extraction of the analytes with acidified acetonitrile and simultaneous liquid-liquid partitioning achieved by an addition of anhydrous magnesium sulfate and sodium acetate without any further clean-up steps. The limits of quantification (LOQs) for α-chaconine and α-solanine were 31 μg kg^?1 and 16 μg kg^?1 of fresh mass, respectively, at the signal-to-noise ratio (S/Ns) of 10. The method was applied in a survey of the content of α-chaconine and α-solanine in twenty commercial potato crisps from different brands. The results showed that all the products contained α-chaconine and α-solanine in widely varying concentrations. The amount of α-chaconine was higher than that of α-solanine in all samples.     

New UPLC–MS/MS method for simultaneous determination of irbesartan and hydrochlorthiazide in human plasma

Ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) is a preeminent analytical tool for rapid biomedical analysis with the objective of reducing analysis time and maintaining good efficiency. In this study a simple, rapid, sensitive and specific ultra-performance liquid chromatography–tandem mass spectrometry method was developed and validated for quantification of the angiotensin II receptor antagonist, irbesartan and hydrochlorthiazide in human plasma. After a simple protein precipitation using methanol and acetonitrile, irbesartan, hydrochlorthiazide and internal standard (IS) telmisartan were separated on Acquity UPLC BEH? C18 column (50 × 2.1 mm, i.d. 1.7 μm, Waters, USA) using a mobile phase consisting of acetonitrile:10 mM ammonium acetate:formic acid (85:15:0.1 % v/v/v) pumped at a flow rate of 0.3 mL/min and detected by tandem mass spectrometry with negative ion mode. The ion transitions recorded in multiple reaction monitoring mode were m/z 427.2 → 193.08 for irbesartan, m/z 295.93 → 268.90 for hydrochlorthiazide and m/z 513.2 → 287.14 for IS. The assay exhibited a linear dynamic range of 30–500 ng/mL for irbesartan and 1–500 ng/mL in human plasma with good correlation coefficient of (0.996) and (0.997) and with a limit of quantitation of 30  and 1 ng/mL for irbesartan and hydrochlorthiazide, respectively. The intra- and inter-assay precisions were satisfactory; the relative standard deviations did not exceed 10.13 % for irbesartan and 11.14 % for hydrochlorthiazide. The proposed UPLC–MS/MS method is simple, rapid and highly sensitive, and hence it could be reliable for pharmacokinetic and toxicokinetic study in both animals and humans.