LC–MS/MS coupled with immunoaffinity extraction for determination of estrone, 17β-estradiol and estrone 3-sulfate in human plasma

Determination of estrogens in plasma is important in evaluation of effects of some anticancer drugs, such as aromatase inhibitors. However, as reported previously, high performance liquid chromatography–radio immunoassay (HPLC–RIA) and liquid chromatography–tandem mass spectrometry (LC–MS/MS) with chemical derivatization require complicated sample preparation. In this study, a highly sensitive and simple method for determination of estrone (E1), 17β-estradiol (E2) and estrone 3-sulfate (E1S) in human plasma has been developed. Following diethylether extraction from plasma, analytes were purified by immunosorbents and then determined by LC–MS/MS using electrospray ionization (ESI). Immunosorbents were prepared by immobilization of specific antibodies raised against each analyte onto solid support. Use of selective immunosorbents in sample preparation removed interference in plasma samples that would cause ionization suppression, and markedly improved the sensitivity of LC–MS/MS for these analytes, without derivatization. Calibration curves of each analyte showed good linearity and reproducibility over the range of 0.05–50 pg/injection for E1, 0.2–50 pg/injection for E2 and 0.05–300 pg/injection for E1S, respectively. The mean values of lower limits of quantification (LLOQ) in human plasma corrected by recovery of deuterated estrogens (internal standard, I.S.) were 0.1892 pg/mL for E1, 0.7064 pg/mL for E2 and 0.3333 pg/mL for E1S, respectively. These LLOQ values were comparable to those previous reported using HPLC–RIA and LC–MS/MS. Using this method, the normal levels of three estrogens in healthy female plasma (n = 5) were determined. The mean values of E1, E2 and E1S were 38.0 pg/mL (range 24.8–53.0), 34.3 pg/mL (22.6–46.6) and 786 pg/mL (163–2080), respectively. The immunoaffinity LC–MS/MS described here allows sensitive and accurate quantification of E1, E2 and E1S without laborious sample preparation.     

Discrepancy between radioimmunoassay and high performance liquid chromatography tandem-mass spectrometry for the analysis of androstenedione

The discrepancy of results for the quantification of androstenedione in human serum between a radioimmunoassay (RIA) method and high performance liquid chromatography tandem-mass spectrometry (LC–MS/MS) was investigated. RIA overestimated concentrations compared to LC–MS/MS on 59 clinical samples (RIA = 1.79 × LC–MS/MS + 0.94). RIA kit and LC–MS/MS calibrants were also determined by both methods. The RIA performed with improved accuracy on the calibrants (RIA = 1.35 × LC–MS/MS − 0.28). Lipid, protein, electrolyte content, and pH of the two sets of calibrants were further investigated. The RIA calibrants contained little lipid material, while the LC–MS/MS calibrant material contained the same levels expected in normal serum/plasma. The pH and sex hormone binding globulin (SHBG) values were different between the RIA calibrants and the LC–MS/MS calibrant material (SHBG, 31 ± 2 and 38 ± 2 nmol/l; pH, 8.27 ± 0.18 and 8.66 ± 0.03, respectively). No correlation was observed between androstenedione RIA and LC–MS/MS discrepancy and lipid or protein. LC–MS/MS sample preparation was tested for the removal of protein-bound material and recovery determined (99–108%). The corresponding RIA results overestimated androstenedione by 52–174% compared to LC–MS/MS. The results here demonstrate that LC–MS/MS is the more accurate method.     

Modified method for determination of sulfur metabolites in plant tissues by stable isotope dilution-based liquid chromatography–electrospray ionization–tandem mass spectrometry

A wide variety of sulfur metabolites play important roles in plant functions. We have developed a precise and sensitive method for the simultaneous measurement of several sulfur metabolites based on liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) and ³⁴S metabolic labeling of sulfur-containing metabolites in Arabidopsis thaliana seedlings. However, some sulfur metabolites were unstable during the extraction procedure. Our proposed method does not allow for the detection of the important sulfur metabolite homocysteine because of its instability during sample extraction. Stable isotope-labeled sulfur metabolites of A. thaliana shoot were extracted and utilized as internal standards for quantification of sulfur metabolites with LC–MS/MS using S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), methionine (Met), glutathione (GSH), and glutathione disulfide (GSSG) as example metabolites. These metabolites were detected using electrospray ionization in positive mode. Standard curves were linear (r² > 0.99) over a range of concentrations (SAM 0.01–2.0 μM, SAH 0.002–0.10 μM, Met 0.05–4.0 μM, GSH 0.17–20.0 μM, GSSG 0.07–20.0 μM), with limits of detection for SAM, SAH, Met, GSH, and GSSG of 0.83, 0.67, 10, 0.56, and 1.1 nM, respectively; and the within-run and between-run coefficients of variation based on quality control samples were less than 8%.     

A new modified Edman procedure for analysis of N-terminal valine adducts in hemoglobin by LC–MS/MS

A rapid and sensitive method using liquid chromatography–tandem mass spectrometry (LC–MS/MS) for simultaneous determination of adducts from acrylamide, glycidamide and ethylene oxide to N-terminal valines in hemoglobin (Hb) was developed. This new procedure is based on the same principles as the N-alkyl Edman procedure for analysis of adducts from electrophilic agents to N-terminal valines in Hb. The N-substituted valines can be detached, enriched and measured selectively as thiohydantoins by the use of an Edman reagent, in this case fluorescein isothiocyanate (FITC). This procedure is denoted as the “adduct FIRE procedure” as the FITC reagent is used for measurement of adducts (R) formed from electrophilic compounds with a modified Edman procedure. In this study, fluorescein thiohydantoin (FTH) analytes of N-substituted valines from acrylamide, glycidamide and ethylene oxide, as well as their corresponding hepta- and tri-deuterium-substituted analogues, were synthesized. These analytes (n = 8) were then characterized by LC–MS/MS (ESI, positive ion mode) and obtained product ions were interpreted. A considerable work with optimization of the FIRE procedure™, resulted in a procedure in which low background levels of the studied adducts could be measured from 250 μL lyzed whole blood samples (human non-smokers). The analytes were enriched and purified with solid phase extraction columns and analyzed by LC–MS/MS with LOQ down to 1 pmol adduct/g Hb. Compared to other procedures for determination of N-terminal Hb adducts, the introduction of FITC has led to a simplified procedure, where whole blood also can be used, giving new opportunities and reduced hand on time with increased sample throughput.     

Improved liquid chromatography–tandem mass spectrometry method for the analysis of eptifibatide in human plasma

A rapid, selective and highly sensitive high performance liquid chromatography–tandem mass spectrometry method (LC–MS/MS) was developed and validated for the determination and pharmacokinetic investigation of eptifibatide in human plasma. Eptifibatide and the internal standard (IS), EPM-05, were extracted from plasma samples using solid phase extraction. Chromatographic separation was performed on a C₁₈ column at a flow rate of 0.5 mL/min. Detection of eptifibatide and the IS was achieved by tandem mass spectrometry with an electrospray ionization (ESI) interface in positive ion mode. Traditional multiple reaction monitoring (MRM) using the transition of m/z 832.6 → m/z 646.4 and m/z 931.6 → m/z 159.4 was performed to quantify eptifibatide and the IS, respectively. The calibration curves were linear over the range of 1–1000 ng/mL with the lower limit of quantitation validated at 1 ng/mL. The intra- and inter-day precisions were within 13.3%, while the accuracy was within ±7.6% of nominal values. The validated LC–MS/MS method was successfully applied for the evaluation of pharmacokinetic parameters of eptifibatide after intravenous (i.v.) administration of a 45 μg/kg bolus of eptifibatide to 8 healthy volunteers.     

Class-specific molecularly imprinted polymers for the selective extraction and determination of sulfonylurea herbicides in maize samples by high-performance liquid chromatography–tandem mass spectrometry

A novel method based on the molecularly imprinted solid-phase extraction (MISPE) procedure has been developed for the simultaneous determination of concentrations of sulfonylurea herbicides such as chlorsulfuron (CS), monosulfuron (MNS), and thifensulfuron methyl (TFM) in maize samples by liquid chromatography–tandem quadrupole mass spectrometry (LC–MS/MS). The molecularly imprinted polymer (MIP) for sulfonylurea herbicides was synthesized by precipitation polymerization using chlorsulfuron as the template molecule, 2-(diethylamino)ethyl methacrylate (DEAMA) as the functional monomer, and trimethylolpropane trimethacrylate (TRIM) as the cross-linker. The selectivities of the chlorsulfuron template and its analogs on the molecularly imprinted polymer were evaluated by high-performance liquid chromatography (HPLC). The extraction and purification procedures for the solid-phase extraction (SPE) cartridge with a molecularly imprinted polymer as the adsorbent for the selected sulfonylurea herbicides were then established. A molecularly imprinted solid-phase extraction method followed by high-performance liquid chromatography–tandem mass spectrometry for the determination of chlorsulfuron, monosulfuron, and thifensulfuron methyl was also established. The mean recoveries of these compounds in maize were in the range 75–110% and the limits of detection (LOD) of chlorsulfuron, monosulfuron, and thifensulfuron methyl were 0.02, 0.75, and 1.45 μg kg⁻¹, respectively. It was demonstrated that the MISPE–HPLC–MS/MS method could be applied to the determination of chlorsulfuron, monosulfuron, and thifensulfuron methyl in maize samples.     

LC–ESI-Q-TOF-MS for faster and accurate determination of microcystins and nodularins in serum

Microcystins (MC) and nodularins (Nod) are cyclic peptide hepatotoxins and tumour promoters produced by cyanobacteria. This study deals with liquid chromatography–mass spectrometry (LC–MS) analyses of 9 major cyanobacterial peptide toxins, starting with a comparison of six small particle size reversed-phase HPLC columns, from which one, Phenomenex Synergi Hydro-RP, was chosen for further chromatography with accurate mass MS studies in a complex biological fluid, serum. The instrumentation used for the serum sample analysis included a Bruker micrO-TOF-Q-MS coupled to an Agilent 1200RR LC system. Total analysis run time per sample was 8.5 min. The Q-TOF-MS instrument was operated on auto MS–MS mode to obtain fragment ions (such as the characteristic fragment m/z 135 from Adda amino acid residue) for toxin identification purposes. Detected mass errors in serum samples were in the range of from 0.3 mDa to 9.1 mDa. The narrow mass window (±20 mDa) for mass chromatograms used in quantitation gave benefits by background noise reduction. We conclude that a LC–ESI-Q-TOF-MS instrumentation is a powerful tool for identification and quantitation of cyanobacterial peptide toxins in a biological matrix.     

Development of a coupled-column liquid chromatographic–tandem mass spectrometric method for the direct determination of betamethasone in urine

Different hyphenated liquid chromatographic (LC) and mass spectrometric (MS) techniques were investigated in order to set-up a method for the fast, direct analysis of betamethasone in hydrolysed and non-hydrolysed urine using large-volume sample injection. After the optimisation of the LC parameters using a traditional UV detector and of the thermospray and mass spectrometric parameters by flow injection, urine samples (0.5 ml) were submitted to analysis by either LC combined with tandem mass spectrometry (MS–MS), coupled-column LC (LC–LC) combined with single quadrupole MS, and LC–LC–MS–MS. Both the three-step configurations (LC–MS–MS and LC–LC–MS) did not provide satisfactory results: loss of sensitivity was noted in the case of LC–MS–MS (likely due to reduced efficiency in the ionisation of betamethasone in the thermospray owing to the presence of large amounts of matrix interference), while in the case of LC–LC–MS a high chemical noise resulting in insufficient selectivity of detection was observed. On the contrary, LC–LC–MS–MS analysis proved to meet the demand of high speed of analysis (sample throughput, 4.5 h⁻¹), selectivity, and sensitivity (LOQ, 1 ng/ml; LOD, 0.2 ng/ml). Notwithstanding the complex analytical system adopted, the developed procedure was manageable and very robust, provided that at the beginning of each analytical session the performance of the system was controlled by checking the retention time of the analytes on the first analytical column with UV detection and by optimising vaporiser temperature of the thermospray by flow injection.     

Identification of metabolites from liquid chromatography–coulometric array detection profiling: Gas chromatography–mass spectrometry and refractionation provide essential information orthogonal to LC–MS/microNMR

Liquid chromatography–coulometric array detection (LC–EC) is a sensitive, quantitative, and robust metabolomics profiling tool that complements the commonly used mass spectrometry (MS) and nuclear magnetic resonance (NMR)-based approaches. However, LC–EC provides little structural information. We recently demonstrated a workflow for the structural characterization of metabolites detected by LC–EC profiling combined with LC–electrospray ionization (ESI)–MS and microNMR. This methodology is now extended to include (i) gas chromatography (GC)–electron ionization (EI)–MS analysis to fill structural gaps left by LC–ESI–MS and NMR and (ii) secondary fractionation of LC-collected fractions containing multiple coeluting analytes. GC–EI–MS spectra have more informative fragment ions that are reproducible for database searches. Secondary fractionation provides enhanced metabolite characterization by reducing spectral overlap in NMR and ion suppression in LC–ESI–MS. The need for these additional methods in the analysis of the broad chemical classes and concentration ranges found in plasma is illustrated with discussion of four specific examples: (i) characterization of compounds for which one or more of the detectors is insensitive (e.g., positional isomers in LC–MS, the direct detection of carboxylic groups and sulfonic groups in ¹H NMR, or nonvolatile species in GC–MS), (ii) detection of labile compounds, (iii) resolution of closely eluting and/or coeluting compounds, and (iv) the capability to harness structural similarities common in many biologically related, LC–EC-detectable compounds.     

Liquid chromatography–tandem mass spectrometry quantification of levosulpiride in human plasma and its application to bioequivalence study

An improved method for determining levels of levosulpiride in human plasma using ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) was developed and validated. The protein precipitation method was used for plasma sample preparation. Levosulpiride and an internal standard (IS) were isocratically separated on a UPLC BEH C₁₈ column with a mobile phase of ammonium formate buffer (1 mM, adjusted to pH 3 with formic acid) and acetonitrile (60:40, v/v). MS/MS detection was performed by monitoring the parent → daughter pair of levosulpiride and the IS at m/z 342 → 112 and 329 → 256, respectively. The method was linear from 2.5 to 200 ng/mL and exhibited acceptable precision and percent recovery. The method was successfully demonstrated in pharmacokinetic and bioequivalence studies of two levosulpiride oral formulations administered to healthy volunteers. When compared to the previous LC–MS methods, the proposed method is faster, well-validated, and uses lesser plasma volume and a similar sensitivity. The use of UPLC allowed rapid and sensitive quantification of levosulpiride, making this method suitable for high-throughput clinical applications.     

Quantitative analysis of glycosaminoglycans,chondroitin/dermatan sulfate,hyaluronic acid,heparan sulfate,and keratan sulfate by liquid chromatography–electrospray ionization–tandem mass spectrometry

We developed a method using liquid chromatography–electrospray ionization–tandem mass spectrometry (LC–ESI–MS/MS) with a selected reaction monitoring (SRM) mode for simultaneous quantitative analysis of glycosaminoglycans (GAGs). Using one-shot analysis with our MS/MS method, we demonstrated the simultaneous quantification of a total of 23 variously sulfated disaccharides of four GAG classes (8 chondroitin/dermatan sulfates, 1 hyaluronic acid, 12 heparan sulfates, and 2 keratan sulfates) with a sensitivity of less than 0.5 pmol within 20 min. We showed the differences in the composition of GAG classes and the sulfation patterns between porcine articular cartilage and yellow ligament. In addition to the internal disaccharides described above, some saccharides derived from the nonreducing terminal were detected simultaneously. The simultaneous quantification of both internal and nonreducing terminal saccharides could be useful to estimate the chain length of GAGs. This method would help to establish comprehensive “GAGomic” analysis of biological tissues.     

Quantitative analysis of 3α,6α,24-trihydroxy-24,24-di(trifluoromethyl)-5β-cholane,a potent synthetic steroidal liver X receptor agonist in plasma using liquid chromatography–tandem mass spectrometry

The steroidal liver X receptor agonist, 3α,6α,24-trihydroxy-24,24-di(trifluoromethyl)-5β-cholane (ATI-829) is a potential therapeutic agent for the treatment of atherosclerosis. A sensitive and selective liquid chromatography–tandem mass spectrometry (LC–MS–MS) method for the quantification of ATI-829 in mouse plasma was developed and validated. Proteins in a 25 μL aliquot of mouse plasma were precipitated, and ATI-829 was extracted from the precipitate by the addition of 125 μL methanol. The overall extraction efficiency was greater than 99%. LC–MS–MS with negative ion electrospray and selected reaction monitoring was used for the quantitative analysis of ATI-829. The lower limit of quantitation of ATI-829 corresponded to 5.0 ng/mL (9.7 nM) plasma. Interference from matrix was negligible. The calibration curve was linear over the range 5–2000 ng/mL. The intra-day precision and inter-day precision of the analyses were <4.5% and <6%, respectively, and the accuracy ranged from 92% to 103%. ATI-829 in plasma was stable for at least 6 h at room temperature, 1 week at 4 °C, and 3 weeks at −20 °C. The validated method was then utilized for pharmacokinetic studies of ATI-829 administered to mice.     

Comparison of fractionation strategies for offline two-dimensional liquid chromatography tandem mass spectrometry analysis of proteins from mouse adipose tissue

In the frame of protein identification from mouse adipose tissue, two strategies were compared for the offline elution of peptides from a strong cation exchange (SCX) column in two-dimensional liquid chromatography tandem mass spectrometry (2D–LC–MS/MS) analyses. First, the salt gradient (using K⁺ as displacing agent) was evaluated from 25 to 500 mM KCl. Then, a less investigated elution mode using a pH gradient (using citric acid and ammonium hydroxide) was carried out from pH 2.5 to 9.0. Equal amounts of peptide digest derived from mouse adipose tissue were loaded onto the SCX column and fractionated according to the two approaches. A total of 15 fractions were collected in two independent experiments for each SCX elution strategy. Then, each fraction was analyzed on a nanoLC–MS/MS platform equipped with a column-switching unit for desalting and enrichment. No substantial differences in peptide quality characteristics (molecular weight, isoelectric point, or GRAVY [grand average of hydropathicity] index distributions) were observed between the two datasets. The pH gradient approach was found to be superior, with 27.5% more unique peptide identifications and 10% more distinct protein identifications compared with the salt-based elution method. In conclusion, our data imply that the pH gradient SCX fractionation is more desirable for proteomics analysis of entire adipose tissue.     

Simultaneous determination of m-nisoldipine and its three metabolites in rat plasma by liquid chromatography–mass spectrometry

A simple, sensitive and selective liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the simultaneous determination of m-nisoldipine and its three metabolites in rat plasma has been developed using nitrendipine as an internal standard (IS). Following liquid–liquid extraction, the analytes were separated using an isocratic mobile phase on a reverse phase C₁₈ column and analyzed by MS in the multiple reaction monitoring (MRM) mode. To avoid contamination by residual sample in the injection syringe, a special injection protocol was developed. We found that m-nisoldipine, metabolite M1 and IS could be ionized under positive or negative electrospray ionization conditions, whereas metabolite M and M2 could only be ionized in the positive mode. The mass spectrometry fragmentation pathways for these analytes are analyzed and discussed herein. The total analysis time required less than 5 min per sample. We employed this method successfully to study the metabolism of m-nisoldipine when it was orally administered to rats at a dose of 9 mg/kg. Three metabolites of m-nisoldipine and an unknown compound of molecular weight 386 were found for the first time in rat plasma. The concentration of the potentially active metabolite was approximately equal to its parent compound concentration.     

Methyl vinyl ketone—Identification and quantification of adducts to N-terminal valine in human hemoglobin

Adducts to N-terminal valines in Hb have been shown useful as biomarkers of exposure to electrophilic compounds. Adducts from many compounds have earlier been measured with a modified Edman degradation method using a GC–MS/MS method. A recently developed method, the adduct FIRE procedure™, adopted for analysis by LC–MS/MS, has been applied in this study. With this method a fluorescein isothiocyanate (FITC) reagent is used to measure adducts (R) from electrophiles with a modified Edman procedure. By using LC–MS/MS in product ion scan mode, a new peak was identified and the obtained MS data indicated that this adduct could originate from methyl vinyl ketone (MVK). Incubation of human-, sheep- and bovine blood with MVK increased the signal of the identified peak. By comparing the LC–MS/MS data from the unknown background peak with data obtained from synthesized fluorescein thiohydantoin (FTH) standards of the MVK adduct to valine and d₈-valine, the identity of this adduct was confirmed. The MVK adduct was shown present in human blood (∼35 pmol/g globin, n = 3) and only just above LOD in bovine blood, n = 1 (LOD = 2 pmol/g globin). MVK reacts, in similarity with acrylamide, via Michael addition. MVK is known to occur in the environment and has earlier been observed in biological samples, which means that there are possible natural and anthropogenic exposure sources. Analysis of an Hb adduct from MVK in humans has to our knowledge not been described before.     

An automated screening method for drugs and toxic compounds in human serum and urine using liquid chromatography–tandem mass spectrometry

A fully automated screening using liquid chromatography–mass spectrometric method applying data-dependent acquisition was developed to identify toxicologically relevant substances in serum and urine. A library including more than 405 spectra of about 365 compounds (main drugs and important metabolites) was established. An easy to use program was created to automate and accelerate library search. Drugs were identified based on their relative retention times, molecular ions and fragment ions. Limits of detection were tested with 100 of the 365 compounds the majority of these were lower than 100 μg/l (67%). The developed LC–MS–MS system seems to be a valuable alternative to other general unknown screening methods allowing fast and specific identification of drugs in serum and urine samples.     

Quantification of corticosteroids in bovine urine using selective solid phase extraction and reversed-phase liquid chromatography/tandem mass spectrometry

This paper presents the development of a simple liquid chromatography–tandem mass spectrometry (LC–MS/MS) method to determine corticosteroids in bovine urine sample matrices. This method uses a single phase extraction (SPE) for cleaning of the sample with an Oasis MAX cartridge at pH 9.0–9.5 and elution by a neutral organic solvent (acetonitrile/dichloromethane), followed by separation on a GEMINI C18 column in the gradient mode with acetate buffer (pH 4.1)/methanol. A triple quadrupole mass spectrometer equipped with a multimode ion source, set to negative atmospheric pressure chemical ionization (APCI) in the multiple reaction monitoring mode was used for detection. The main advantage of this method over other commonly used methods includes the use of SPE with a low volume cartridge for sample preparation and no ion suppression effects from matrix components of the urine samples in the LC–MS/MS analysis. This allowed a reduction the quantification limits (decision limits, CCα) for the first time to 0.1 μg/L (1 and 0.2 μg/L for triamcinolone and flumethasone, respectively). The developed method was validated in accordance with the European Union Commission Decision 2002/657 EC. The recoveries and within-laboratory reproducibility varied from 77% to 115% and 87% to 107.5%, respectively, at 2, 3, and 4 μg/L levels of corticosteroids. The relative standard deviation (RSD) of the measurements was lower than 30%. The decision limit was calculated by multiplying the signal-to-noise ratio by 3 and the obtained values were in the range of 0.1–1.0 μg/L, confirmed by the analysis of twenty blank samples, which were spiked at the desired concentrations. The detection capability was calculated by the addition of the decision limit and the standard deviation followed by multiplication by 1.64 of the within-laboratory reproducibility at 2 μg/L of corticosteroids. The method was applied to four urine samples, giving concentrations of prednisolone (PRED) residues in the range from 0.3 to 0.9 μg/L.     

Determination of vinpocetine and its primary metabolite,apovincaminic acid,in rat plasma by liquid chromatography–tandem mass spectrometry

A precise and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for simultaneous determination of vinpocetine (VP) and its primary metabolite, apovincaminic acid (AVA), in rat plasma was developed and validated. The analytes and the internal standard-dimenhydrinate were extracted from 50 μL aliquots of rat plasma via solid–liquid extraction. Chromatographic separation was achieved in a run time of 3.5 min on a C₁₈ column under isocratic conditions. Detection of analytes and IS was done by tandem mass spectrometry, operating in positive ion and multiple reaction monitoring (MRM) acquisition mode. The protonated precursor to product ion transitions monitored for VP, AVA and IS were m/z 351.4 → 280.2, 323.2 → 280.2 and 256.2 → 167.3 respectively. The method was fully validated for its sensitivity, selectivity, accuracy and precision, matrix effect, stability study and dilution integrity. A linear dynamic range of 0.5–500 ng/mL for both VP and AVA was evaluated with mean correlation coefficient (r) of 0.9970 and 0.9984 respectively. The precision of the assay (RSD%) was less than 8.55% at all concentrations levels for both VP and AVA. This method was successfully applied to a pharmacokinetic study of VP in rats after intravenous (1 mg/kg) and oral (1 mg/kg) administration.     

Combining recombinant ribonuclease U2 and protein phosphatase for RNA modification mapping by liquid chromatography–mass spectrometry

Ribonuclease (RNase) mapping of modified nucleosides onto RNA sequences is limited by RNase availability. A codon-optimized gene for RNase U2, a purine selective RNase with preference for adenosine, has been designed for overexpression using Escherichia coli as the host. Optimal expression conditions were identified enabling generation of milligram-scale quantities of active RNase U2. RNase U2 digestion products were found to terminate in both 2′,3′-cyclic phosphates and 3′-linear phosphates. To generate a homogeneous 3′-linear phosphate set of products, an enzymatic approach was investigated. Bacteriophage lambda protein phosphatase was identified as the optimal enzyme for hydrolyzing cyclic phosphates from RNase U2 products. The compatibility of this enzymatic approach with liquid chromatography–tandem mass spectrometry (LC–MS/MS) RNA modification mapping was then demonstrated. RNase U2 digestion followed by subsequent phosphatase treatment generated nearly 100% 3′-phosphate-containing products that could be characterized by LC–MS/MS. In addition, bacteriophage lambda protein phosphatase can be used to introduce ¹⁸O labels within the 3′-phosphate of digestion products when incubated in the presence of H₂¹⁸O, allowing prior isotope labeling methods for mass spectrometry to include digestion products from RNase U2.