Development and validation of a LC/MS/MS method for 6-keto PGF1α, a metabolite of prostacyclin (PGI₂)

A sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed for the quantitation of 6-keto PGF_1α in human urine and plasma. Prostacyclin (PGI₂) is a locally acting prostanoid, which mediates vasorelaxation and inhibition of platelet aggregation. 6-Keto PGF_1α is the most-immediate metabolite of PGI₂.Samples were spiked with an internal standard (6-keto PGF_1α-d₄), purified by immuno-affinity chromatography and selected reaction monitoring (SRM) was performed.Analytical validation of the 6-keto PGF_1α assay was performed in urine. This included an assessment of assay precision, recovery, stability, sensitivity and linearity. Urinary 6-keto PGF_1α concentrations were also correlated to urinary 2,3-dinor-6-keto PGF_1α (PGIM) concentrations using urine samples collected from 16 healthy volunteers. The mean concentration of 6-keto PGF_1α in urine (mean ± SD) was 92 ± 51 pg/ml or 168 ± 91 pg/mg creatinine. Overall, there was a statistically significant correlation between urinary 6-keto PGF_1α and PGIM (r² = 0.55, p ≤ 0.001; slope = 2.7; y-intercept = 130). However, PGIM was approximately 3-fold more abundant than 6-keto PGF_1α in urine. In addition, 6-keto PGF_1α concentrations were measured in EDTA plasma samples obtained from 7 healthy donors. The mean concentration of 6-keto PGF_1α in plasma was 1.9 ± 0.8 pg/ml (±SD).     

HPLC–MS and HPLC–MS/MS analysis of seven active constituents of Xiao-Xu-Ming decoction and application to a pharmacokinetic study after oral administration to rat

Xiao-xu-ming decoction (XXMD) is a traditional Chinese medicine that has been widely used to treat theoplegia and its sequelae. This paper reports the development of three separate assays based on reversed phase high-performance liquid chromatography–mass spectrometry (HPLC–MS) and HPLC–MS/MS for the determination of seven active constituents of XXMD viz oroxylin A-7-O-glucuronide, wogonoside, liquiritigenin, cimifugin, 5-O-methylvisammiol, glycyrrhizic acid and glycyrrhetinic acid in rat plasma. All calibration curves were linear (r >0.99) with lower limits of quantitation (LLOQs)<12.4 ng/mL. Intra- and inter-day precisions (as relative standard deviation) were all <10.7% with recoveries in the range of 88.7–113%. In addition, the seven analytes were shown to be stable in rat plasma samples under relevant storage conditions. The validated methods were successfully applied to a pharmacokinetic study in rat after oral administration of XXMD.     

Determination of cepharanthine in rat plasma by LC–MS/MS and its application to a pharmacokinetic study

Context: Cepharanthine (CPA) has been reported to possess a wide range of pharmacological activities.

Objective: This study investigates the pharmacokinetic characteristics after oral or intravenous administration of CPA by using a sensitive and rapid LC–MS/MS method.

Materials and methods: A sensitive and rapid LC–MS/MS method was developed for the determination of CPA in Sprague–Dawley rat plasma. Twelve rats were equally randomized into two groups, including the intravenous group (1?mg/kg) and the oral group (10?mg/kg). Blood samples (250?μL) were collected at designated time points and determined using this method. The pharmacokinetic parameters were calculated.

Results: The calibration curve was linear within the range of 0.1–200?ng/mL (r?=?0.999) with the lower limit of quantification at 0.1?ng/mL. After 1?mg/kg intravenous injection, the concentration of CPA reached a maximum of 153.17?±?16.18?ng/mL and the t_1/2 was 6.76?±?1.21?h. After oral administration of 10?mg/kg of CPA, CPA was not readily absorbed and reached C_max 46.89?±?5.25?ng/mL at approximately 2.67?h. The t_1/2 was 11.02?±?1.32?h. The absolute bioavailability of CPA by oral route was 5.65?±?0.35%, and the bioavailability was poor.

Discussion and conclusions: The results indicate that the bioavailability of CPA was poor in rats, and further research should be conducted to investigate the reason for its poor bioavailability and address this problem.     

Determination of oxaceprol in rat plasma by LC–MS/MS and its application in a pharmacokinetic study

A sensitive method for the quantification of oxaceprol in rat plasma using high-performance liquid chromatography–tandem mass spectrometry (LC–MS/MS) was developed. Sample pretreatment involved a simple protein precipitation by the addition of 60 μL of acetonitrile–methanol (1:2, v/v) to 20 μL plasma sample volume. Separation was achieved on a Dikma ODS-C18 (5 μm, 150 mm × 4.6 mm) reversed-phase column at 40 °C with acetonitrile/0.1% formic acid–4 mM ammonium acetate in water (35:65,v/v) at a flow rate of 0.6 mL/min. Detection was performed using an electrospray ionization (ESI) operating in negative ion multiple reaction monitoring (MRM) mode by monitoring the ion transitions from m/z 172 → 130 (oxaceprol) and m/z 153 → 109 (protocatechuic acid, internal standard). The calibration curve of oxaceprol in plasma showed good linearity over the concentration range of 1.25–800 ng/mL. The limit of detection and limit of quantification were 0.400 ng/mL and 1.25 ng/mL, respectively. Intra- and inter-day precisions in all samples were within 15%. There was no matrix effect. The validated method was successfully applied to a preclinical pharmacokinetic study of oxaceprol in rats. After oral administration of 20 mg/kg oxaceprol to rats, the main pharmacokinetic parameters T_max, C_max, T_1/2, V_z/F and AUC_0–t were 1.4 h, 1.2 μg/mL, 2.3 h, 19.7 L/kg and 3.4 mg h/L, respectively.     

Development and validation of a highly sensitive LC–MS/MS method for the determination of acacetin in human plasma and its application to a protein binding study

A highly sensitive bioanalytical method for the quantification of acacetin in human plasma was developed and comprehensively validated using liquid chromatography-tandem mass spectrometry (LC–MS/MS). A minimal volume of human plasma sample (20 μL) was prepared by simple deproteinization with 80 μL of acetonitrile. Chromatographic separation was performed using Kinetex C₁₈ column with an isocratic mobile phase consisting of water and acetonitrile (20:80, v/v) containing 0.1 % formic acid at a flow rate of 0.3 mL/min over a total run time of 2.0 min. Mass spectrometric detection was performed using multiple reaction-monitoring modes at the mass/charge transitions m/z 285.22 → 242.17 for acacetin and m/z 277.59 → 175.04 for chlorpropamide (internal standard). The calibration curve was linear over the range of 0.1–500 ng/mL with a lower limit of quantitation of 0.1 ng/mL. The coefficients of variation for both intra- and inter-day validation were less than 11.9 %, and the intra- and inter-day accuracy ranged from 96.8 to 108 %. Mean recovery of acacetin in human plasma was within the range of 91.5–95.6 %. This validated LC–MS/MS method was successfully applied to a human plasma protein binding study that indicated extensive and concentration-independent protein binding of acacetin in human plasma.     

Biopharmaceutical and pharmacokinetic characterization of matrine as determined by a sensitive and robust UPLC–MS/MS method

The purpose of this research was to develop a sensitive and reproducible UPLC–MS/MS method to analyze matrine, an anticancer compound, and to use it to investigate its biopharmaceutical and pharmacokinetic behaviors in rats. A sensitive and fast UPLC–MS/MS method was successfully applied to determine matrine in rat plasma, intestinal perfusate, bile, microsomes, and cell incubation media. The absolute oral bioavailability of matrine is 17.1 ± 5.4% at a dose of 2 mg/kg matrine. Matrine at 10 μM was shown to have good permeability (42.5 × 10⁻⁶ cm/s) across the Caco-2 cell monolayer, and the ratio of P_A–B to P_B–A was approximately equal to 1 at two different concentrations (1 and 10 μM). Perfusion study showed that matrine displayed significant differences (P < 0.05) in permeability at different intestinal regions. The rank order of permeability was ileum (highest, P_w = 6.18), followed by colon (P_w = 2.07), duodenum (P_w = 0.61) and jejunum (P_w = 0.52). Rat liver microsome studies showed that CYP and UGTs were not involved in matrine metabolism. In conclusion, a sensitive and reliable method capable of measuring matrine in a variety of matrixes was developed and successfully used to determine absolute oral bioavailability of matrine in rats, transport across Caco-2 cell monolayers, absorption in rat intestine, and metabolism in rat liver microsomes.     

Development of a simultaneous LC–MS/MS method to predict in vivo drug–drug interaction in mice

Cocktail substrates are useful in investigating drug–drug interactions (DDI) that can rapidly identify the cytochrome P450 (CYP) isoforms that interact with test drugs. In this study, we developed and validated five probe drugs for CYP1A, CYP2B, CYP2C, CYP2D, and CYP3A using LC–MS/MS to determine CYP activities in mice. The five probe substrates were caffeine (2 mg/kg), bupropion (30 mg/kg), omeprazole (4 mg/kg), dextromethorphan (40 mg/kg), and midazolam (2 mg/kg) for CYP1A, CYP2B, CYP2C, CYP2D, and CYP3A, respectively. The cocktail substrates were orally administered to male 5-week-old ICR mice over 0–240 min. The analytical method was validated; it showed high selectivity, linearity, and acceptable accuracy. We confirmed the lack of interaction of this cocktail in the control state (no effect of CYP inducer or inhibitor) and suggested AUC_ratio (metabolite/substrate) as a unit to evaluate DDI in vivo. In addition, the cocktail assay was applied for the determination of pharmacokinetic parameters against phenobarbital as a selective CYP2B inducer and ketoconazole as a strong CYP3A inhibitor. The concentration of cocktail substrates and the LC–MS/MS method were optimized. In conclusion, we developed a simultaneous and comprehensive analysis system for predicting potential DDI in mice.     

Validated LC–MS/MS assay for the determination of felbinac: Application to a preclinical pharmacokinetics study of felbinac trometamol injection in rat

A rapid and sensitive analytical method based on high-performance liquid chromatography–tandem mass spectrometry (LC–MS/MS) has been developed for the determination of felbinac in rat plasma, bile, urine, feces and tissue. Sample preparation involved liquid–liquid extraction with ethyl ether–dichloromethane (60:40, v/v). Chromatography of felbinac and the internal standard probenecid was performed within 2 min on a Venusil MP C₁₈ column (100 mm × 4.6 mm i.d., 5 μm) with a mobile phase consisting of acetonitrile–5 mM ammonium acetate containing 0.1% formic acid (pH 3.0) (80:20, v/v) at a flow rate of 1.2 ml/min. Detection by electrospray negative ionization mass spectrometry and multiple-reaction monitoring of the transitions of felbinac at m/z 211.1 → 167.0 and of probenecid at m/z 283.9 → 239.9 was linear over the concentration range 5–5000 ng/ml with a lower limit of quantitation of 5 ng/ml using a sample volume of only 50 μl. Intra- and inter-day precisions (as relative standard deviation, R.S.D.) were ≤7.3% and ≤6.4%, respectively, and accuracy (as relative error, R.E.) was in the range −2.1 to 7.4%. Recoveries and matrix effects were satisfactory in all the biological matrices examined. The method was applied to a preclinical pharmacokinetic study in rat involving a single intravenous injection of felbinac trometamol.     

Development and validation of a rapid and sensitive UHPLC–MS/MS method for the determination of paliperidone in beagle dog plasma

In order to evaluate the pharmacokinetic profile of paliperidone extended-release tablets in vivo, a simple and rapid ultra-high performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) method was developed and validated for the determination of paliperidone in beagle dog plasma. Paliperidone and diazepam (internal standard) were extracted from plasma samples with diethyl ether, and then separated on a C₁₈ column (2.1 × 50 mm, 2.6 μm) under gradient elution with methanol–0.1% formic acid at a flow rate of 0.3 ml/min. The compounds were detected using a triple-quadrupole mass spectrometer equipped with an electrospray ionization (ESI) source. The validated method was linear over the concentration range of 1.00–1000.00 ng/ml and the lower limit of quantitation was 1.00 ng/ml. The intra-day and inter-day precision values were not more than 15% (relative standard deviation < 20% at low levels), while the accuracy was within ±10% of nominal values. The validated UHPLC–MS/MS method was successfully applied to an oral pharmacokinetic study of paliperidone extended-release tablets in a beagle dog.     

UPLC–MS/MS for the determination of azilsartan in beagle dog plasma and its application in a pharmacokinetics study

The purpose of the study is to develop an ultra performance liquid chromatography-tandem mass spectrometry (UPLC–MS/MS) to determinate the concentration of azilsartan in the dog plasma. After precipitated by methanol, the plasma sample containing azilsartan and diazepam (internal standard, IS) was determined by UPLC–MS/MS. The mobile phase consisted of acetonitrile-water was pumped at a flow rate of 0.3 ml/min in gradient elution. Kinetex 2.6 μ XB-C18 column (50 × 2.1 mm, 100 Å; Phenomenex, USA) were used for LC separations. The column temperature was 30 °C and the injection volume was 5 μl. The electrospray ionization (ESI) and multiple reaction monitoring (MRM) were applied at the transitions of m/z 457 → 279 (azilsartan) and m/z 285 → 193 (diazepam), respectively. The developed method was identified a good linearity over a concentration range of 2.5–5000 ng/ml. The lower limit of quantitation (LLOQ) was 2.5 ng/ml. The intra-day and inter-day precision (relative standard deviation, RSD%) were less than 10% and accuracy (relative error, RE %) was less than 5% at three quality control levels. The extraction recovery of azilsartan at three quality control levels were 82.41 ± 0.68%, 98.66 ± 11.00%, 102.43 ± 0.82%. And the recovery for IS (100 ng/ml) was 91.75 ± 0.54%. A validated UPLC–MS/MS method was firstly developed for the quantification of azilsartan in dog plasma and it was applied to the pharmacokinetics study.     

UPLC–MS/MS assay for quantification of an inhibitor of kinases (Foretinib) in plasma: Application to a pharmacokinetic study in rats

Foretinib, an oral multikinase inhibitor, is known to have anti-tumor effects against cancers. The doses and the levels of foretinib vary based on the type of cancer to be treated. An accurate and precise method is required to determine the level of foretinib and its pharmacokinetics. Here, we developed such a method, which was validated based on the guidelines of the FDA and EMA. Foretinib and ibrutinib (the internal standard (IS)) were extracted using tert-butyl methyl ether. Foretinib and IS were eluted in approximately 1.2 min. Thus, a linear, fast, accurate, and precise method was developed. The calibration curve was linear (r² ˃ 0.997) in the range of 0.5–400.0 ng/mL and the lowest limit of quantitation was 0.5 ng/mL. The average recovery, accuracy, and precision were 87.9%, 88.7%, and ≤7.8%, respectively. The analyte was deemed stable using various stability tests. The validated assay was then fruitfully applied to a pharmacokinetics study in rats, which revealed that foretinib was absorbed and the maximum concentration achieved at 4.0 h after the administration of a single dose of foretinib.     

Aflatoxins contamination in Pakistani brown rice: a comparison of TLC,HPLC, LC–MS/MS and ELISA techniques

Advancement in the field of analytical food-chemistry has explored various experimental techniques for aflatoxins (AFs) quantification. The present study was aimed to compare four different techniques; thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), liquid chromatography–tandem mass spectrometry (LC–MS/MS) and enzyme-linked immunosorbent assay (ELISA) for the analysis of aflatoxin B₁ (AFB₁), B₂ (AFB₂), G₁ (AFG₁) and G₂ (AFG₂) in brown rice (n?=?120) being collected from Karachi, Pakistan. All the four assays provide precised, accurate and comparable results. However, some differences were observed. For instance, TLC, HPLC and LC–MS/MS methodologies offered the advantage of the quantification of individual toxins in contrast to ELISA technique. The contamination ranges of AFB₁/AFB₂ as determined by TLC, HPLC and LC–MS/MS were 1.18–9.97/0.59–1.52, 0.16–10.54/0.26–1.35 and 0.11–10.88/0.38–1.48?µg/kg, respectively. However, AFG₁ and AFG₂ were not detected in any tested samples. Furthermore, owing to low-detection limit and sensitivity, HPLC and LC–MS/MS methodologies have identified greater number of contaminated samples in comparison to TLC and ELISA techniques. The overall average results of total AFs as provided by HPLC (3.79?µg/kg) and LC–MS/MS (3.89?µg/kg) were found higher in comparison to TLC (3.68?µg/kg) and ELISA (3.70?µg/kg). On the basis of achieved results, it was concluded that TLC, HPLC, LC–MS/MS and ELISA techniques are valuable tool for the quantification of AFs in cereals and grains. Furthermore, HPLC and LC–MS/MS techniques offer an added advantage for the detection of AFs in diminutive levels.     

Separation and characterization of forced degradation products of abacavir sulphate by LC–MS/MS

Abacavir sulphate was subjected to forced degradation under the conditions of hydrolysis (acid, alkali and neutral), oxidation, photolysis and thermal stress as prescribed by ICH. Eight degradation products were formed and their separation was accomplished on Waters XTerra C₁₈ (250 mm × 4.6 mm, 5 μm) column using 20 mM ammonium acetate:acetonitrile as a mobile phase in gradient elution mode by LC. The degradation products were characterized by LC–MS/MS and its fragmentation pathways were proposed. No previous reports were found in the literature regarding the degradation behavior of abacavir sulphate.     

Determination of tramadol and metabolites by HPLC-FL and HPLC–MS/MS in urine of dogs

Tramadol is a centrally acting analgesic drug used in veterinary and human clinical practice. Its metabolism has been largely characterized in human being but is still long to be comprehended in several animal species, especially in the dog. The aim of the present study was to develop and validate a new analytical procedure to investigate HPLC the metabolization/elimination process tramadol in urine of dogs by HPLC-FL or HPLC–MS/MS. A single oral dose of tramadol (4 mg/kg) was administered to 4 male Beagle dogs and the urine was naturally collected. This matrix either hydrolyzed than un-hydrolyzed was extracted with different blends of solvents to detect the total or free form of the analytes, respectively.     

LC–MS/MS studies of ritonavir and its forced degradation products

Forced degradation of ritonavir (RTV), under the conditions of hydrolysis (acidic, basic and neutral), oxidation, photolysis and thermal stress as prescribed by ICH was studied using LC–MS/MS. Eight degradation products were formed and their separation was accomplished on Waters XTerra^® C₁₈ column (250 mm × 4.6 mm i.d., 5 μm) using water:methanol:acetonitrile as (40:20:40, v/v/v) mobile phase in an isocratic elution mode by LC. The method was extended to LC–MS/MS for characterization of the degradation products and the pathways of decomposition were proposed. No previous reports were found in the literature regarding the characterization of degradation products of ritonavir.     

Development of a LC–MS/MS-based method for determining metolazone concentrations in human plasma: Application to a pharmacokinetic study

In this study, a method was developed and validated for the quantification of metolazone in human plasma samples. This method involves high-performance liquid chromatography coupled with tandem mass spectrometry and is more sensitive, selective and rapid than currently available methods. Chromatography was performed using a Phenomenex^® Luna C18 column (100 mm × 2.0 mm, 5 μm, 100 Å) with an isocratic mobile phase of 0.1% formic acid:acetonitrile (40:60, v/v) and zaleplon as an internal standard. The drug and internal standard were extracted by liquid–liquid extraction and analyzed by mass spectrometry in the multiple reaction monitoring mode by using m/z values of 366.20/259.10 for metolazone and 306.20/235.60 for zaleplon. The calibration curve was linear over metolazone concentrations ranging from 0.02 ng/mL to 15 ng/mL. The lower limit of quantification was 0.02 ng/mL. Intra- and inter-assay precisions were 0.9–4.8% and 4.2–6.3%, respectively. The intra- and inter-assay accuracies in quantifying metolazone were 97.5–102.3% and 99.2–104.0%, respectively. Metolazone and zaleplon were eluted within 3.6 minutes, and the retention time was 1.75 minutes for metolazone and zaleplon. The validated method was successfully applied to a pharmacokinetic study of metolazone in human plasma.     

LC–NMR and LC–MS identification of an impurity in a novel antifungal drug icofungipen

Successful use of LC–NMR and LC–MS for rapid identification of an impurity in a novel antifungal drug icofungipen has been demonstrated. Complementary information obtained from the two methods made it possible to determine the structure of A1 prior to its isolation and purification. Stop-flow LC–NMR (¹H and DQFCOSY), LC–MS and LC–MS/MS spectra have shown that A1 is structurally related to icofungipen. It was later isolated and prepared synthetically and its structure was corroborated by high-resolution NMR spectroscopy.     

Development of a LC–MS/MS method for the determination of antrodin B and antrodin C from Antrodia camphorata extract in rat plasma for pharmacokinetic study

A selective and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated for the determination of antrodin B and antrodin C in rat plasma. Both target compounds, together with the internal standard (diazepam), were extracted from rat plasma samples by liquid–liquid extraction with ethyl acetate. Chromatographic separation was carried out on an Agilent XDB-C₈ column with an isocratic mobile phase consisting of acetonitrile and water (70:30, V/V) at a flow rate of 0.5 mL/min. The mass spectrometric detection was performed by selected reaction monitoring (SRM) mode via atmospheric pressure chemical ionization (APCI) source operating in positive ionization mode. The assay exhibited a linear dynamic range of 47.6–4760 ng/mL for antrodin B and 56.6–5660 ng/mL for antrodin C. The intra- and inter-day precision was less than 5.3% and the accuracy was less than 2.7% for both analytes. The validated method has been applied to the pharmacokinetic study of antrodin B and antrodin C in rats following oral administration of Antrodia camphorata extract.     

Characterization of identity, metabolism and androgenic activity of 17-hydroxyandrosta-3,5-diene by GC–MS and a yeast transactivation system

Anabolic–androgenic steroids are frequently misused compounds in sports, and they belong to the controlled substances according to the requirements of the World Anti-Doping Agency. The classical techniques of steroid detection are mass spectrometry coupled to gas or liquid chromatography. Biological methods that base on the ability of substances to bind the steroid receptor are not applied in routine doping control procedures so far, but they appear to be useful for characterization of steroid androgenic potential. In this study we used the yeast androgen receptor reporter system (YAS), which in the past has already successfully been applied to both various androgenic substances and also urine samples. Giving attention to the androgenic potential of steroidal dietary supplements, we exemplified the analysis using both mass spectrometry techniques and the YAS-based assay on the product “Syntrax Tetrabol” which was a confiscated dietary supplement and marketed as a steroid precursor. Identification, structure and the kinetic behavior of its excreted metabolites were analyzed by NMR, GC–MS and LC–MS/MS. The androgenic potential of the parent compound as well as its metabolites in urine was evaluated with the help of the YAS. The application of urine samples with a previous deconjugation and the inclusion of urine density values were carried out and led to increased responses on the YAS. Further, the possibility of a complementary application of structure-based instrumental analysis and biological detection of androgenicity with the help of the YAS seems to be desirable and is discussed.     

SAR and LC/MS studies of β-lactamic inhibitors of human fatty acid amide hydrolase (hFAAH): evidence of a nonhydrolytic process

The endocannabinoid hydrolyzing enzyme FAAH uses a nonclassical catalytic triad (namely, Ser-Ser-Lys instead of Ser-Asp-His) to cleave its endogenous substrates. Because inhibiting FAAH has a clear therapeutic potential, we previously developed β-lactam-type inhibitors of hFAAH. Here, we report the synthesis of five novel derivatives (5-9) of our lead compound 1-(pent-4-enoyl)-3(S)-[1(R)-(4-phenylbutanoyloxy)-ethyl]-azetidin-2-one (4, IC(50) = 5 nM) obtained via the systematic replacement of one to three carbonyls by methylene groups. The SAR results showed that the imide, but not the lactam, function is essential to the inhibition of hFAAH. We also performed LC/MS analysis following incubation of our inhibitors with hFAAH or mouse liver. We demonstrated that hFAAH interacts with these β-lactam-type inhibitors but, unexpectedly, does not open the β-lactam moiety. This mechanism seems to be unique to FAAH because the β-lactam function of the inhibitors is hydrolyzed when they are incubated in the presence of the serine hydrolases expressed in the mouse liver. Finally, we confirmed these results by showing that a highly selective FAAH inhibitor (PF-750) does not prevent this hydrolysis by liver homogenates.