Validation and implementation of a method for determination of bryostatin 1 in human plasma by using liquid chromatography/tandem mass spectrometry

A new sensitive and specific method using liquid chromatography/tandem mass spectrometry for determination of bryostatin 1 was developed and validated. Sample pretreatment involved a double liquid-liquid extraction step with a mixture of acetonitrile/n-butyl chloride (1/4, v/v). Separation of the compound of interest, including the internal standard paclitaxel, was achieved on a Waters X-Terra C18 (50 x 2.1 mm i.d., 3.5 microm) analytical column with acetonitrile/water mobile phase (80:20, v/v) containing 0.1% formic acid using isocratic flow at 0.15 mL/min for 13 min. The analytes of interest were monitored by tandem mass spectrometry with electrospray positive ionization. The linear calibration curves were generated over the range of 50-2000 pg/mL with values for the coefficient of determination of >0.99. The values for both within-day and between-day precision and accuracy were <15%. This method was used to characterize the plasma pharmacokinetics of bryostatin 1 at doses of 20 microg/m2) to optimize treatment with this agent.     

Determination of casopitant and its three major metabolites in dog and rat plasma by positive ion liquid chromatography/tandem mass spectrometry

A sensitive, selective and quantitative method for the simultaneous determination of casopitant, a potent and selective antagonist of the human Neurokinin 1 (NK-1) receptor, and its three major metabolites M12, M13 and M31 was developed and validated in dog and rat plasma. Acetonitrile containing stable labeled internal standards for the four analytes was used to precipitate proteins in plasma. Chromatographic separation was obtained using a reversed phase column with multiple reaction monitoring turboionspray positive ion detection. The lower and upper limits of quantification for casopitant and its metabolites were 15 and 15,000 ng/mL, using a 50 μL of dog or rat plasma aliquot, respectively. The inter-day precision (relative standard deviation) and accuracy (relative error) in dog plasma, derived from the analysis of validation samples at 5 concentrations, ranged from 4.1% to 10.0% and −10.8% to 8.7%, respectively, for casopitant and its 3 major metabolites. The intra-day precision (relative standard deviation) and accuracy (relative error) in rat plasma, derived from the analysis of validation samples at 5 concentrations, ranged from 3.9% to 6.6% and −9.6% to 8.3%, respectively, for casopitant and its three metabolites. All analytes were found to be stable in analytical solutions for at least 43 days at 4 °C, in dog and rat plasma at room temperature for at least 24 h, at the storage temperature of −20 °C for at least 6 months, and following the action of three freeze–thaw cycles from −20 °C to room temperature. All analytes were also found to be stable in processed extracts at 4 °C for at least 72 h. This assay proved to be accurate, precise, fast and was used to support long-term toxicology studies in dog and rat.     

Development of a reverse-phase liquid chromatography electrospray ionization mass spectrometry method for lipidomics, improving detection of phosphatidic acid and phosphatidylserine

In the studies of lipid metabolomics, liquid chromatography electrospray ionization mass spectrometry (LC/MS) is a robust and popular technique. Although effective reverse-phase LC methods enabling the separation of phospholipid molecular species have been developed, there are still problems with the separation of phosphatidic acid (PA) and phosphatidylserine (PS). These acidic phospholipids often elute as extensively broad peaks, causing inferior separation, detection, and quantification-a severe limitation of the method. In this study, we have developed reverse-phase LC conditions that reduce the undesired peak tailings in the elution profiles of both PA and PS, by using a starting mobile phase containing a low concentration of phosphoric acid (5 microM) and a high percentage of water (40%). Our method sensitively analyzes PA, PS, and their lysoforms, as well as the other phospholipids within a biological sample, in a single chromatographic step by an LC/MS method and, thus, is suitable for lipidomics.     

Development and validation of a sensitive liquid chromatography/mass spectrometry method for quantitation of flavopiridol in plasma enables accurate estimation of pharmacokinetic parameters with a clinically active dosing schedule

A high-performance liquid chromatographic assay with tandem mass spectrometric detection was developed and validated for quantitation of the broad spectrum kinase inhibitor, flavopiridol, in human plasma. Sample preparation conditions included liquid-liquid extraction in acetonitrile (ACN), drying, and reconstitution in 20/80 water/ACN. Flavopiridol and the internal standard (IS), genistein, were separated by reversed phase chromatography using a C-18 column and a gradient of water with 25 mM ammonium formate and ACN. Electrospray ionization and detection of flavopiridol and genistein were accomplished with single reaction monitoring of m/z 402.09>341.02 and 271.09>152.90, respectively in positive-ion mode [M+H](+) on a triple quadrupole mass spectrometer. Recovery was greater than 90% throughout the linear range of 3-1000 nM. Replicate sample analysis indicated within- and between-run accuracy and precision to be less than 13% throughout the linear range. This method has the lowest lower limit of quantitation (LLOQ) reported to date for flavopiridol, and it allows for more accurate determination of terminal phase concentrations and improved pharmacokinetic parameter estimation in patients receiving an active dosing schedule of flavopiridol.     

Potential bias and mitigations when using stable isotope labeled parent drug as internal standard for LC-MS/MS quantitation of metabolites

In recent years, increasing emphasis has been placed on quantitative characterization of drug metabolites for better insight into the correlation between metabolite exposure and toxicological observations or pharmacological efficacy. One common strategy for metabolite quantitation is to adopt the stable isotope labeled (STIL) parent drug as the internal standard in an isotope dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay. In the current work, we demonstrate this strategy could have a potential pitfall resulting in quantitation bias if the internal standard is subject to ion suppression from the co-eluting parent drug in the incurred samples. Propranolol and its metabolite 4-hydroxypropranolol were used as model compounds to demonstrate this phenomenon and to systematically evaluate different approaches to mitigate the issue, including atmospheric pressure chemical ionization (APCI) mode of ionization, increased internal standard concentration, quantitation without internal standard, the use of a structural analog as internal standard, and dilution of the samples. Case studies of metabolite quantitation in nonclinical and clinical studies in drug development were also included to demonstrate the importance of using an appropriate bioanalytical strategy for metabolite quantitation in the real world. We present that bias of metabolite concentrations could pose a potential for poor estimation of safety risk. A strategy for quantitation of metabolites in support of drug safety assessment is proposed.     

Identification and quantification of peptides and proteins secreted from prostate epithelial cells by unbiased liquid chromatography tandem mass spectrometry using goodness of fit and analysis of variance

The proteins secreted by prostate cancer cells (PC3(AR)6) were separated by strong anion exchange chromatography, digested with trypsin and analyzed by unbiased liquid chromatography tandem mass spectrometry with an ion trap. The spectra were matched to peptides within proteins using a goodness of fit algorithm that showed a low false positive rate. The parent ions for MS/MS were randomly and independently sampled from a log-normal population and therefore could be analyzed by ANOVA. Normal distribution analysis confirmed that the parent and fragment ion intensity distributions were sampled over 99.9% of their range that was above the background noise. Arranging the ion intensity data with the identified peptide and protein sequences in structured query language (SQL) permitted the quantification of ion intensity across treatments, proteins and peptides. The intensity of 101,905 fragment ions from 1421 peptide precursors of 583 peptides from 233 proteins separated over 11 sample treatments were computed together in one ANOVA model using the statistical analysis system (SAS) prior to Tukey-Kramer honestly significant difference (HSD) testing. Thus complex mixtures of proteins were identified and quantified with a high degree of confidence using an ion trap without isotopic labels, multivariate analysis or comparing chromatographic retention times.     

Development and validation of a high-throughput method for the quantitative analysis of d-amphetamine in rat blood using liquid chromatography/MS on a hybrid triple quadrupole-linear ion trap mass spectrometer and its application to a pharmacokinetic study

Amphetamines are a group of sympathomimetic drugs that exhibit strong central nervous system stimulant effects. d-Amphetamine ((+)-alpha-methylphenetylamine) is the parent drug in this class to which all others are structurally related. In drug discovery, d-amphetamine is extensively used either for the exploration of novel mechanisms involving the catecholaminergic system, or for the validation of new behavioural animal models. Due to this extensive use of d-amphetamine in drug research and its interest in toxicologic–forensic investigation, a specific and high-throughput method, with minimal sample preparation, is necessary for routine analysis of d-amphetamine in biological samples. We propose here a sensitive, specific and high-throughput bioanalytical method for the quantitative determination of d-amphetamine in rat blood using MS³ scan mode on a hybrid triple quadrupole-linear ion trap mass spectrometer (LC–MS/MS/MS). Blood samples, following dilution with water, were prepared by fully automated protein precipitation with acetonitrile containing an internal standard. The chromatographic separation was achieved on a Waters XTerra C18 column (2.1 mm × 30 mm, 3.5 μm) using gradient elution at a flow rate of 1.0 mL/min over a 2 min run time. An Applied Biosystems API4000 QTRAP™ mass spectrometer equipped with turbo ion-spray ionization source was operated simultaneously in MS³ scan mode for the d-amphetamine and in multiple reaction monitoring (MRM) for the internal standard. The MS/MS/MS ion transition monitored was m/z 136.1 → 119.1 → 91.1 for the quantitation of d-amphetamine and for the internal standard (rolipram) the MS/MS ion transition monitored was m/z 276.1 → 208.2. The linear dynamic range was established over the concentration range 0.5–1000 ng/mL (r² = 0.9991). The method was rugged and sensitive with a lower limit of quantification (LLOQ) of 0.5 ng/mL. All the validation data, such as accuracy, precision, and inter-day repeatability, were within the required limits. This method was successfully applied to evaluate the pharmacokinetics of d-amphetamine in rat. On a more general extent, this work demonstrated that the selectivity of the fragmentation pathway (MS³) can be used as alternative approach to significantly improve detection capability in complex situation (e.g., small molecules in complex matrices) rather than increasing time for sample preparation and chromatographic separation.     

Development and validation of a liquid chromatography/tandem mass spectrometry assay for the simultaneous determination of nateglinide, cilostazol and its active metabolite 3,4-dehydro-cilostazol in Wistar rat plasma and its application to pharmacokinetic study

Nateglinide (NTG), an insulin secretogogue, has been studied in rats for drug-drug interaction with cilostazol (CLZ), an antiplatelet agent commonly used in diabetics. We developed a liquid chromatography tandem mass spectrometry (LC-MS/MS) based method that is capable of simultaneous monitoring plasma levels of nateglinide, cilostazol, and its active metabolite 3,4-dehydro-cilostazol (DCLZ). All analytes including the internal standard (Repaglinide) were chromatographed on reverse phase C(18) column (50 mm x 4.6mm i.d., 5 microm) using acetonitrile: 2mM ammonium acetate buffer, pH 3.4 (90:10, v/v) as mobile phase at a flow rate 0.4 ml/min in an isocratic mode. The detection of analyte was performed on LC-MS/MS system in the multiple reaction monitoring (MRM) mode. The quantitations for analytes were based on relative concentration. The method was validated over the concentration range of 20-2000 ng/ml and the lower limit of quantitation was 20 ng/ml. The recoveries from spiked control samples were >79% for all analytes and internal standard. Intra- and inter-day accuracy and precision of validated method were with in the acceptable limits of <15% at all concentration. The quantitation method was successfully applied for simultaneous estimation of NTG, CLZ and DCLZ in a pharmacokinetic drug-drug interaction study in Wistar rats.     

Development and validation of a sensitive ultra performance liquid chromatography tandem mass spectrometry method for the analysis of fentanyl and its major metabolite norfentanyl in urine and whole blood in forensic context

Fentanyl and its major metabolite norfentanyl often occur in low doses in biological samples. Therefore, a highly sensitive liquid chromatography tandem mass spectrometry (LC–MS/MS) method has been developed and fully validated. Sample preparation was performed on a mixed-mode cation exchange solid phase extraction (SPE) cartridge with an additional alkaline wash step to decrease matrix effects and thus increase sensitivity. Ionization of fentanyl and norfentanyl with electrospray ionization (ESI) was more efficient than atmospheric pressure chemical ionization (APCI). The use of a mobile phase of high pH resulted in higher ESI signals than the conventional low pH mobile phases. In the final method, gradient elution with 10 mM ammonium bicarbonate (pH 9) and methanol was performed. A comparison of columns with different internal diameter and/or smaller particles showed optimal resolution and sensitivity when an Acquity C18 column (1.7 μm, 2.1 mm × 50 mm) was used. Deuterium labeled internal standards were used, but with careful evaluation of their stability since loss of deuteriums was seen. With limits of detection of 0.25 pg/ml for fentanyl and 2.5 pg/ml for norfentanyl in urine and 5 pg/ml for fentanyl and norfentanyl in whole blood the presented method is highly appropriate for the analysis of fentanyl and norfentanyl in forensic urine and blood samples.     

Detection and quantitation of N-(deoxyguanosin-8-yl)-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine adducts in DNA using online column-switching liquid chromatography tandem mass spectrometry

The heterocyclic aromatic amine, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is formed by the grilled cooking of certain foods such as meats, poultry and fish. PhIP has been shown to induce tumours in the colon, prostate and mammary glands of rats and is regarded as a potential human dietary carcinogen. PhIP is metabolically activated via cytochrome P450 mediated oxidation to an N-hydroxylamino-PhIP intermediate that is subsequently converted to an ester by N-acetyltransferases or sulfotransferases and undergoes heterolytic cleavage to produce a PhIP-nitrenium ion, which reacts with DNA to form the N-(deoxyguanosin-8-yl)-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP-C8-dG) adduct. Thus far, the detection and quantification of PhIP-DNA adducts has relied to a large extent on ³²P-postlabelling methodologies. In order to expand the array of available techniques for the detection and improved quantification of PhIP-C8-dG adducts in DNA we have developed an online column-switching liquid chromatography (LC)–electrospray ionization (ESI)-tandem mass spectrometry (MS/MS) selected reaction monitoring (SRM) method incorporating an isotopically [¹³C₁₀]-labelled PhIP-C8-dG internal standard for the analysis of DNA enzymatically hydrolysed to 2′-deoxynucleosides. A dose-dependent increase was observed for PhIP-C8-dG adducts when salmon testis DNA was reacted with N-acetoxy-PhIP. Analysis of DNA samples isolated from colon tissue of mice treated by oral gavage daily for 5 days with 50 mg/kg body weight of PhIP resulted in the detection of an average level of 14.8 ± 3.7 PhIP-C8-dG adducts per 10⁶ 2′-deoxynucleosides. The method required 50 μg of hydrolysed animal DNA on column and the limit of detection for PhIP-C8-dG was 2.5 fmol (1.5 PhIP-C8-dG adducts per 10⁸ 2′-deoxynucleosides). In summary, the LC–ESI-MS/MS SRM method provides for the rapid automation of the sample clean up and a reduction in matrix components that would otherwise interfere with the mass spectrometric analysis, with sufficient sensitivity and precision to analyse DNA adducts in animals exposed to PhIP.     

Serum steroid profiling by isotopic dilution-liquid chromatography-mass spectrometry: comparison with current immunoassays and reference intervals in healthy adults

Background

The simultaneous, rapid and reliable measurement of a wide steroid panel is a powerful tool to unravel physiological and pathological hormone status. Clinical laboratories are currently dominated by high-throughput immunoassays, but these methods lack specificity due to cross-reactivity and matrix interferences. We developed and validated an isotopic dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) method for the simultaneous measurement of cortisol, corticosterone, 11deoxycortisol, androstenedione, deoxycorticosterone (DOC), testosterone, 17OHprogesterone, dehydroepiandrosterone (DHEA) and progesterone in serum, and compared it to routine immunoassays employed in our laboratory. We also established adult reference intervals in 416 healthy subjects.

Methods

0.9 ml of serum were spiked with labelled internal standards (IS) and extracted on C18 cartridges. Eluate was injected into a two-dimensional LC-system, purified in a perfusion column and separated on a C8 column during a 21 min gradient run. Analytes were revealed by atmospheric pressure chemical ionization (APCI) followed by multiple reaction monitoring (MRM) analysis.

Results

Of the four immunoassays compared with the ID-LC-MS/MS method, only the results of ElecsysE170 for cortisol, testosterone in males and progesterone > 1 ng/ml were in agreement with ID-LC-MS/MS. ElecsysE170 for testosterone in females and progesterone < 1 ng/ml, Immulite2000 for androstenedione, DSL-9000 for DHEA and 17OHP Bridge for 17OHprogesterone, respectively, showed poor agreement. Reference intervals and steroid age and fertility related fluctuations were established.

Conclusion

Our ID-LC-MS/MS method proved to be reliable and sensitive in revealing steroid circulating concentrations in adults and in highlighting the limits of routine immunoassays at low concentrations.