UPLC-MS/MS quantification of nanoformulated ritonavir, indinavir, atazanavir, and efavirenz in mouse serum and tissues

Animal pharmacokinetic and tissue distribution assays of antiretroviral therapeutic drugs require accurate drug quantification in biological fluids and tissues. Here we report a simple, rapid, and sensitive ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for quantification of commonly used antiretroviral drugs ritonavir (RTV), indinavir (IDV), atazanavir (ATV), and efavirenz (EFV) in mouse serum and tissues (liver, kidney, lung, and spleen). These antiretroviral drugs are currently the cornerstones of common therapeutic regimens for human immunodeficiency virus (HIV) infection. Chromatographic separation was achieved using a gradient mobile phase (5% acetonitrile in methanol and 7.5mM ammonium acetate (pH 4.0)) on an ACQUITY UPLC(?)BEH Shield RP 18 column. All compounds eluted within a 7 min run time. Lopinavir was used as an internal standard. Detection was achieved by dual positive and negative ionization modes on a quadrupole linear ion trap hybrid mass spectrometer with an electrospray ionization (ESI) source. The dynamic range was 0.2-1000 ng/mL for RTV, IDV, and ATV, and 0.5-1000 for EFV. The method was validated and showed high and consistent intra-day and inter-day accuracy and precision for all analytes. This method is used to support the preclinical development studies of targeted- and sustained-release combination ART (nanoART). The current data demonstrate a 1.5-4 fold increase in serum and tissue AUC of nanoformulated ATV, RTV, and EFV administered to mice when compared to native drug. In addition, the tested formulation enhanced exposure of the same anti-HIV drugs in mouse tissues.     

Quantitative determination of cyclic phosphatidic acid in human serum by LC/ESI/MS/MS

An LC/ESI/MS/MS method for cyclic phosphatidic acid (cPA) quantification in serum is established in the present report. The limit of quantitation of the assay reaches low nanomolar level in human serum and the CV% are within 10%. Using this method, we successfully quantify the levels of two cPA species, 16:0 and 18:1, in human serum. We find that the concentrations of 16:0 cPA in the serum of normal subjects and post-surgery ovarian cancer patients are significantly higher than its corresponding concentration in pre-surgery ovarian cancer patients, supporting the observation that cPA has anti-cancer activity. Another discovery is that the addition of strong acids (such as hydrochloric acid) in human serum may lead to the production of artificial cPA. Therefore, strong acids should be avoided in the extraction of cPA present in a complex matrix. Based on this observation, a new lipid extraction method was developed and used to extract cPA. The extraction recovery is close to 80%, guaranteeing an accurate quantification of cPA by LC/ESI/MS/MS can be performed.     

Free amino acid quantification by LC-MS/MS using derivatization generated isotope-labelled standards

The further development of derivatizing reagents for plasma amino acid quantification by tandem mass spectrometry is described. The succinimide ester of 4-methylpiperazineacetic acid (MPAS), the iTRAQ reagent, was systematically modified to improve tandem mass spectrometer (MS/MS) product ion intensity. 4-Methylpiperazinebutyryl succinimide (MPBS) and dimethylaminobutyryl succinimide (DMABS) afforded one to two orders of magnitude greater MS/MS product ion signal intensity than the MPAS derivative for simple amino acids. CD(3) analogues of the modified derivatizing reagents were evaluated for preparation of amino acid isotope-labelled quantifying standards. Acceptable accuracy and precision was obtained with d(3)-DMABS as the amino acid standards derivatizing reagent. The product ion spectra of the DMABS amino acid derivatives are diagnostic for structural isomers including valine/norvaline, alanine/sarcosine and leucine/isoleucine. Improved analytical sensitivity and specificity afforded by these derivatives may help to establish liquid chromatography tandem mass spectrometry (LC-MS/MS) with derivatization generated isotope-labelled standards a viable alternative to amino acids analysers.     

Highly sensitive quantification of 7alpha-hydroxy-4-cholesten-3-one in human serum by LC-ESI-MS/MS

We describe a highly sensitive and specific method for the quantification of serum 7alpha-hydroxy-4-cholesten-3-one (C4), which has been used as a biomarker for bile acid biosynthesis. This method is based upon a stable isotope dilution technique by liquid chromatography-tandem mass spectrometry (LC-MS/MS). C4 was extracted from human serum (2-50 mul) by a salting-out procedure, derivatized into the picolinoyl ester (C4-7alpha-picolinate), and then purified using a disposable C(18) cartridge. The resulting picolinoyl ester derivative of C4 was quantified by LC-MS/MS using the electrospray ionization mode. The detection limit of the C4 picolinoyl ester was found to be 100 fg (signal-to-noise ratio = 10), which was approximately 1,000 times more sensitive than the detection limit of C4 with a conventional HPLC-ultraviolet method. The relative standard deviations between sample preparations and between measurements by our method were calculated to be 5.7% and 3.9%, respectively, by one-way layout analysis. The recovery experiments were performed using serum spiked with 20.0-60.0 ng/ml C4 and were validated by a polynomial equation. The results showed that the estimated concentration with 95% confidence limit was 23.1 +/- 2.8 ng/ml, which coincided completely with the observed X(0) +/- SD = 23.3 +/- 1.0 ng/ml with a mean recovery of 93.4%. This method provides highly reliable and reproducible results for the quantification of C4, especially in small volumes of blood samples.     

Rapid quantification of free and esterified phytosterols in human serum using APPI-LC-MS/MS

A novel analytical platform based on liquid chromatography and tandem mass spectrometry using atmospheric pressure photoionization was applied for the simultaneous quantification of free and esterified beta-sitosterol, campesterol, brassicasterol, and stigmasterol. The total time for sample pretreatment and analysis could be reduced from approximately 3 h [gas chromatography-mass spectrometry (GC-MS)] to 15 min. The detection limits of the different phytosterols ranged between 0.25 and 0.68 microg/l. Linear ranges were between 1 and 1,000 microg/l. The within-run and between-run variabilities ranged between 1.4% and 9.9%. The analytical sensitivity was at least 150-fold higher compared with GC-MS. Our new method allows a rapid and simultaneous determination of free and esterified phytosterols in serum.     

Identification and Quantification of Several Mammalian Steroid Hormones in Plants by UPLC-MS/MS

We have developed an effective method for the isolation, identification, and quantification of several mammalian steroid hormones and their metabolites in different plant tissues. The purification protocol was based on solid-phase extraction (SPE) combined with immunoaffinity chromatography (IAC) using immobilized generic polyclonal anti-Δ⁴-3-keto-steroid antibodies covalently bound to Affi-Gel 10 sorbent. The antibodies were characterized by means of enzyme-linked immunosorbent assay (ELISA). The detection limit of the ELISA was 6.0 × 10⁻¹⁰ mol L⁻¹ and cross-reactivity with most Δ⁴-3-keto-steroids was very high as predicted (68–122%). The IAC allowed fast, single-step purification of different plant extracts prior to analysis by ultra-performance liquid chromatography-electrospray tandem mass spectrometry [UPLC-ESI(+)-MS/MS]. In multiple-reaction-monitoring (MRM) mode, the detection limit of the method for most of the steroids analyzed was close to 10 fmol and the response was linear up to 50 pmol injected. The analytical accuracy was validated using tobacco leaf samples spiked with known amounts of authentic and deuterium-labeled standards. The newly developed method was capable of detecting and quantifying at least 12 specified steroid compounds in plant extracts. In the analyzed extracts from three plant species, that is, common foxglove (Digitalis purpurea L.), tobacco (Nicotiana tabacum L.), and elecampane inula (Inula helenium L.), four endogenous steroids were detected, identified, and quantified. Progesterone was found in all three plants at concentrations comparable to those reported in previous studies. Three other steroids, androstendione, 17α-hydroxyprogesterone, and 16-dehydroprogesterone, were identified for the first time in plant extracts. 17α-Hydroxyprogesterone and 16-dehydroprogesterone occurred at significant concentrations in D. purpurea, whereas androstendione was found in N. tabacum and I. helenium but not in D. purpurea.     

Simultaneous quantification of cholesterol sulfate,androgen sulfates,and progestagen sulfates in human serum by LC-MS/MS

Steroids are primarily present in human fluids in their sulfated forms. Profiling of these compounds is important from both diagnostic and physiological points of view. Here, we present a novel method for the quantification of 11 intact steroid sulfates in human serum by LC-MS/MS. The compounds analyzed in our method, some of which are quantified for the first time in blood, include cholesterol sulfate, pregnenolone sulfate, 17-hydroxy-pregnenolone sulfate, 16-α-hydroxy-dehydroepiandrosterone sulfate, dehydroepiandrosterone sulfate, androstenediol sulfate, androsterone sulfate, epiandrosterone sulfate, testosterone sulfate, epitestosterone sulfate, and dihydrotestosterone sulfate. The assay was conceived to quantify sulfated steroids in a broad range of concentrations, requiring only 300 μl of serum. The method has been validated and its performance was studied at three quality controls, selected for each compound according to its physiological concentration. The assay showed good linearity (R² > 0.99) and recovery for all the compounds, with limits of quantification ranging between 1 and 80 ng/ml. Averaged intra-day and between-day precisions (coefficient of variation) and accuracies (relative errors) were below 10%. The method has been successfully applied to study the sulfated steroidome in diseases such as steroid sulfatase deficiency, proving its diagnostic value. This is, to our best knowledge, the most comprehensive method available for the quantification of sulfated steroids in human blood.     

MS/MS library facilitated MRM quantification of native peptides prepared by denaturing ultrafiltration

ABSTRACT: Naturally occurring native peptides provide important information about physiological states of an organism and its changes in disease conditions but protocols and methods for assessing their abundance are not well-developed. In this paper, we describe a simple procedure for the quantification of non-tryptic peptides in body fluids. The workflow includes an enrichment step followed by two-dimensional fractionation of native peptides and MS/MS data management facilitating the design and validation of LC- MRM MS assays. The added value of the workflow is demonstrated in the development of a triplex LC-MRM MS assay used for quantification of peptides potentially associated with the progression of liver disease to hepatocellular carcinoma.     

Development and validation of a LC/MS/MS method for simultaneous quantification of oxcarbazepine and its main metabolites in human serum

A fast, sensitive and specific LC/MS/MS method for the simultaneous analysis of oxcarbazepine (OXC), 10-hydroxycarbazepine (MHD) and trans-diol-carbazepine (DHD), in human serum, has been developed and validated. Serum drugs were extracted by C8 solid-phase cartridges (SPE) and separated in less than 3 min on a C18 reverse-phase column using an isocratic elution. A tandem mass spectrometer, as detector, was used for quantitative analysis in positive mode by a multiple reaction monitoring. Calibration curves, obtained on two ranges of concentration (0.78-50 mg/L for MHD and 0.078-5.0 mg/L for OXC and DHD), showed correlation coefficients (r) better than 0.997. Within day and between days quality controls imprecision, as CV%, ranged from 0.3 to 4.6% and from 1.9 to 5.8%, respectively. Cyheptamide (CYE) was used as internal standard. No detectable carry-over and no relevant cross-talk and matrix effect occurred. Samples from 24 treated patients were analysed and drug serum concentrations obtained by this method are in agreement with those of other methods and also are well correlated (r=0.88) in comparison to our routine HPLC-UV method. Based on the analytical results and short run time, the method is suitable to support routine analysis of therapeutic drugs monitoring from human serum of treated patients or for pharmacokinetic studies.     

A Rapid Determination of 30 Prohibited Pesticide Residues in Lycii Fructus by UPLC-MS/MS

Prohibited pesticide residues have become one of the main factors affecting the quality and safety of Lycii Fructus, However, rarely studies focus on the rapid determination of these residues. Here, a total of 30 kinds of prohibited pesticide residues were determined by ultra-performance liquid chromatography tandem triple quadrupole mass spectrometry (UPLC-MS/MS) in five different process ways. Pretreatment methods, chromatographic separation and detection conditions in mass spectrometry were all optimized accordingly. Among the five different pretreatment methods, the first and third solid phase extraction failed to provide high recoveries of sulfosulfuron compounds (both lower than 60%). Recovery of chlorphenamidine by the Quick Easy Cheap Effective Rugged and Safe multiresidue method (QuEChERS) was lower than 60%, which did not meet the requirements of trace determination. The concentrations of 30 prohibited pesticides residues treated by straightforward and solid phase extraction showed good linearity in their corresponding ranges, with correlation coefficients over 0.99. The average recoveries of straightforward ranged from 78.13% to 110.9%, while RSD ranged from 1.3% to 16.9%, albeit poor purification was observed. The recovery yield from solid phase extraction was between 67.75% and 103.08% with RSD value from 0.8% to 14.0%, which met the requirements of trace determination, this method has good precision and stability. These results could be employed to other Traditional Chinese Medicines (TCMs) in detecting prohibited pesticide residues.     

Inhibition of the isoprenoid biosynthesis pathway; detection of intermediates by UPLC-MS/MS

The isoprenoid biosynthesis pathway provides the cell with a variety of compounds which are involved in multiple cellular processes. Inhibition of this pathway with statins and bisphosphonates is widely applied in the treatment of hypercholesterolemia and metabolic bone disease, respectively. In addition, since isoprenylation of proteins is an important therapeutic target in cancer research there is interest in interfering with isoprenoid biosynthesis, for which new inhibitors to block farnesylation and geranylgeranylation of small GTPases are being developed. We recently developed a sensitive method using UPLC-MS/MS that allows the direct detection and quantification of all intermediates of the mevalonate pathway from MVA to GGPP which can be used to verify the specificity of inhibitors of the isoprenoid biosynthesis pathway. We here investigated the specificity of several inhibitors of the isoprenoid biosynthesis pathway in HepG2 cells, fibroblasts and lymphoblasts. The nitrogen-containing bisphosphonates pamidronate and zoledronate specifically inhibit farnesyl pyrophosphate synthase indicated by the accumulation of IPP/DMAPP. However, zaragozic acid A, a squalene synthase inhibitor, causes an increase of MVA in addition to the expected increase of FPP. Analysis of isoprenoid intermediate profiles after incubation with 6-fluoromevalonate showed a very nonspecific result with an increase in MVA, MVAP, MVAPP and IPP/DMAPP. These results show that inhibitors of a particular enzyme of the isoprenoid biosynthesis pathway can have additional effects on other enzymes of the pathway either direct or indirect through accumulation of isoprenoid intermediates. Our method can be used to test new inhibitors and their effect on overall isoprenoid biosynthesis.     

Multiplex quantification of lamprey specific bile acid derivatives in environmental water using UHPLC-MS/MS

Larval and adult sea lampreys (Petromyzon marinus) release bile salts and acids into the surrounding aquatic environment. Some of these bile salts and acids, such as petromyzonol sulfate (PZS), 3-keto petromyzonol sulfate (3k PZS), petromyzonamine disulfate (PADS), petromyzosterol disulfate (PSDS), and 3-keto allocholic acid (3k ACA), may function as pheromones. To examine the release and distribution patterns of these metabolites, which this study has termed bile acid derivatives, we developed a novel UHPLC-MS/MS method that was characterized by simple sample preparation, baseline separation, and short analysis time for all studied compounds. These five analytes were separated in 7 min using a reversed-phase C18 column containing 1.7 μm particles and a gradient elution at pH 8.9. Once separated, the analytes were subjected to electrospray ionization-mass spectrometry (negative ion mode) and collision-induced dissociation tandem mass spectrometry (CID-MS/MS) using the multiple reaction monitoring (MRM) mode. Deuterated 3k PZS ([(2)H(5)]3k PZS) was added as the internal standard (IS) to the sample prior to solid phase extraction (SPE). Among the three types of SPE sorbent tested, mixed-mode cation-exchange and reversed-phase sorbent for bases (MAX) and acids (MCX), and reversed-phase C18 sorbent (Sep-pak), the best recoveries (84.1-99.7%) were obtained with MCX cartridges. The calibration curves of all five analytes were linear between 0.15 and 1200 ng/mL, with R(2)≥0.9997. This method had a precision of relative standard deviation (RSD) ≤9.9% and an accuracy of deviation (DEV) ≥92.5%. The developed method was successfully used to quantify bile acid derivatives found in streams where lampreys spawn (SD<1.4) and water conditioned with male sea lampreys (SD<4.8). Utilizing this method provides a routine analysis of lamprey bile acid derivatives and may prove useful for sea lamprey population estimates in future studies and applications.     

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.     

Simple and sensitive method for quantification of low tobramycin concentrations in human plasma using HPLC-MS/MS

After oral administration of tobramycin, as part of selective decontamination of the digestive tract (SDD) in critically ill patients, absorption of tobramycin from the gut into the blood may take place. To quantify low concentrations of tobramycin in human plasma, we developed and validated a simple (sample pre-treatment consisting of protein precipitation with acetonitrile using 200microl plasma), rapid (runtime 3min using a Pathfinder MR reversed-phase column) and sensitive (concentration range of 0.05-1.0mg/l using MS/MS detection) method.     

Sensitive quantification of sirolimus and everolimus by LC–MS/MS with online sample cleanup

Sirolimus and its derivative everolimus are widely used today as immunosuppressive agents for example in the transplantation medicine. The problematic pharmacokinetic behavior of those substances makes therapeutic drug monitoring mandatory. Therefore, a fast, simple and sensitive high-throughput procedure using online extraction with turbulent flow chromatography for the concurrent measurement of sirolimus and everolimus has been developed. 200 μl of whole blood was mixed with internal standard (23-desmethoxyrapamycin) and the precipitation solution and centrifuged. An aliquot of the supernatant was transferred into autosampler vials. 50 μl of the supernatant was injected into the LC system, where the analytes were extracted using turbulent flow chromatography and thereafter analyzed using reversed phase chromatography. Detection was done by atmospherical pressure chemical ionization (APCI) mass spectrometry in the negative ionization mode. The method has been fully validated and compared to a previously used method. The method was shown to be linear over the entire calibration range (2.2–43.7 μg/l for everolimus and 2.9–51.2 μg/l for sirolimus). The lower limit of quantification was 0.5 μg/l for both compounds. For within-day and between-day analysis, the CV's were <7.6% for everolimus and <8.7% for sirolimus, respectively. The accuracy was between 92.1% and 105% for everolimus and 96.1% and 106% for sirolimus. Recovery ranged between 46.3% and 50.6% for everolimus and 51.2% and 57.2% for sirolimus. The method was demonstrated to be free of matrix effects and comparable to the previously used method. The presented LC–MS/MS method, using turbulent flow chromatography online extraction, allows a fast, simple and reliable determination of everolimus and sirolimus.     

Absolute quantification of the model biomarker prostate-specific antigen in serum by LC-Ms/MS using protein cleavage and isotope dilution mass spectrometry

Protein cleavage-isotope dilution mass spectrometry (PC-IDMS) can be used to quantify proteins, with an isotope-labeled analogue of the peptide fragment used as an internal standard. Here, we investigate use of a standard LC-MS/MS platform for quantifying a model biomarker directly from serum by this technique. We synthesized a peptide (IVGGWECEK) identical to the N-terminal tryptic fragment of PSA but with each glycine containing two 13C atoms and one 15N atom. PSA-free human serum was denatured with urea followed by the introduction of PSA standard and the stable isotope labeled internal standard peptide. The sample was then proteolyzed with trypsin and subjected to quantification using LC-MS/ MS on a triple quadrupole mass spectrometer. A linear least squares calibration curve made from five different concentrations of PSA added to serum and digested (each made in triplicate and randomly injected three times) had a mean slope of 0.973 (SE = 0.023), intercept of -0.003 (SE = 0.022), and R2 of 0.971. Recovery of calibrators ranged from 70 to 85% with a mean run-to-run CV of 13% and a mean within-run CV of 5.7%. PC-IDMS is a promising technique for quantifying proteins covering a broad range of applications from standardizing immunoassays to monitoring post-translational modifications to quantifying newly discovered biomarkers prior to the development and implementation of an immunoassay, just to name a few. Issues surrounding the application of PC-IDMS for the absolute quantification of proteins include selection of a proteolytic fragment for quantification that can be cleaved and isolated reproducibly over a broad dynamic range, stable isotope labeled synthetic peptide standards that give consistent results, and LC-MS/MS methods that provide adequate sensitivity and reproducibility without creating impractical analysis times. The results presented here show that absolute quantification can be performed on the model biomarker PSA introduced into denatured serum when analyzed by LC-MS/MS. However, concerns still exist regarding sensitivity compared to existing immunoassays as well as the reproducibility of PC-IDMS performed in different matrixes.     

Traceability of Amanita fuliginea poisoning using DNA barcoding and UPLC-MS/MS

Amanita fuliginea is a lethal poisonous mushroom found in Japan and southern China. The primary toxins are α-amanitin (α-AMA) and β-amanitin (β-AMA). There is a lack of systematic and comprehensive investigations on the traceability of A. fuliginea poisoning due to technological limitations. This study aimed to examine whether A. fuliginea poisoning incidents could be traced using DNA barcoding and ultraperformance liquid chromatography coupled with triple quadrupole mass spectrometry (UPLC-MS/MS). We collected A. fuliginea specimens and prepared cooked and cooked plus simulated gastric fluid (SGF)-treated samples. We then performed DNA barcoding of internal transcribed spacer regions for species identification and UPLC-MS/MS for toxin level determination. Our results indicate that under the experimental conditions used herein, DNA barcoding can be used for molecular identification of mushroom samples that are cooked and/or cooked plus SGF-treated for less than 30 min; UPLC-MS/MS can be used for toxin analysis of cooked and SGF-treated (0–1440 min) samples. This is the first time that DNA barcoding and UPLC-MS/MS have been combined for studying the toxicological traceability of A. fuliginea using simulated gastric contents or vomit in northern China. Our data provide support for the treatment of clinical mushroom poisoning cases.     

Identification and quantification of components in extracts of Uncaria tomentosa by HPLC-ES/MS

The two main classes of secondary metabolites, alkaloids and quinovic acid glycosides, of Uncaria tomentosa (Willd.) DC. (Rubiaceae), a Peruvian plant commonly known as ‘uña de gato’, have been analysed. Separation of the alkaloidal fraction was achieved using a solid phase extraction method based on cationic exchange, and an analytical method employing HPLC‐ES/MS has been developed. Quantitative data for commercial wild bark, cultivated bark and leaves are reported. The analysis of quinovic acid glycosides was performed directly on the crude extract using both a fast analytical method based on ?ow injection ES/MS, and a more complete analytical technique using HPLC‐MS. Copyright © 2004 John Wiley & Sons, Ltd.