Validated Concentration Range Research Articles

Dried blood spot analysis for the quantification of vancomycin and creatinine using liquid chromatography – tandem mass spectrometry: Method development and validation

BackgroundVancomycin is a widely used antibiotic for the treatment of gram-positive bacterial infections, especially for methicillin-resistant Staphylococcus aureus (MRSA) infections. Due to a small therapeutic range and large inter-patient variability, therapeutic drug monitoring (TDM) of vancomycin is required to minimize toxicity and maximize treatment efficacy. Venous blood sampling is mostly applied for TDM of vancomycin, although this widely used sampling method is more invasive compared to less painful alternatives, such as the dried blood spot (DBS) method, which can be performed at home. MethodWe developed an UPLC-MS/MS method for the quantification of vancomycin and creatinine in DBS. A fast sample preparation and short analysis run time of 5.2 min were applied, which makes this method highly suitable for clinical settings. Validation was performed according to international (FDA and EMA) guidelines. ResultsThe validated concentration range was found linear for creatinine from 41.8 µmol/L to 722 µmol/L and for vancomycin from 3.8 mg/L to 76.6 mg/L (r2 > 0.990) and the inaccuracies, imprecisions, hematocrit effects, and recoveries were < 15 % for both compounds. No significant carryover effect was observed. ConclusionHence, we successfully validated a quantification method for the simultaneous determination of creatinine and vancomycin in DBS.

LC-MS/MS quantitation of non-psychotropic cannabinoid cannabidiol in aqueous humor

Cannabidiol (CBD) is the most abundant non-psychotropic phytocannabinoid isolated from Cannabis sativa. To support preclinical studies of ocular pharmacology of CBD, a bioanalytical method was developed and validated for quantification of CBD in aqueous humor using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Aqueous humor samples were subjected to protein precipitation by acetonitrile, followed by chromatographic separation using reversed phase LC on a Raptor ARC-18 column with mobile phase A: 0.1 % (v/v) formic acid in water (B) 0.1 % formic acid in acetonitrile (B) as eluents. Detection was carried out with a triple quadrupole mass spectrometer with electrospray ionization operated in positive ion mode. Stable-isotope labeled CBD (CBD-d3) was used as internal standard. The total run time was 8 min. Quantification was accomplished within the validated concentration range of 0.5–500 ng/mL for CBD using a 5 µL sample. The lower limit of quantitation was 0.5 ng/mL. Inter- and intra-day precision is 4.737–7.620 % and 3.426–5.830 %, respectively. Inter- and intra-day accuracy ranged between 99.01 % and 100.2 % and 99.85–101.4 % respectively. The extraction recoveries were found to be 66.06 ± 5.146 %. The established method was successfully applied to investigate ocular pharmacokinetics of CBD in mice. Following intraperitoneal (i.p.) administration of 50 mg/kg CBD, its concentration reaches a Cmax of 71.55 ± 36.64 ng/mL in aqueous humor, with a Tmax of 2 h and a half-life of 1.046 h. The AUC was 183.4 ± 49.17 ng * h/mL. The development and validation of this LC-MS/MS method is an important step toward the goal of assessing the aqueous humor concentrations of CBD and correlating the concentrations of this phytocannabinoid with its ocular pharmacologic effects.

A sensitive liquid chromatographic-mass spectrometry method for the quantification of vincristine in whole blood collected with volumetric absorptive microsampling

Vincristine is a well-established cytotoxic drug. In paediatric populations blood collection via venipuncture is not always feasible. Volumetric absorptive microsampling (VAMS) is a less invasive method for blood collection. Furthermore, VAMS lacks the haematocrit effect on the recovery known with dried blood spots. Therefore, a liquid chromatography tandem-mass spectrometry method was developed and validated for the quantification of vincristine in whole blood collected with VAMS devices. Sample preparation consisted of solid-liquid extraction with 0.2% formic acid in water and acetonitrile. The final extract was injected on a C18 column (2.0 ×50 mm, 5 µm). Gradient elution was used and quantification was accomplished with a triple quadruple mass spectrometer operating in the positive mode. The validated concentration range was from 1 to 50 ng/mL with an intra- and inter-accuracy and precision of ± 10.3% and ≤ 7.3%, respectively. This method was able to successfully quantify vincristine concentrations in whole blood collected with VAMS from paediatric oncology patients. Vincristine concentrations in whole blood were non-linearly associated with plasma concentrations, which could be described with a saturable binding equilibrium model.

On-Line Solid Phase Extraction High Performance Liquid Chromatography Method Coupled With Tandem Mass Spectrometry for the Therapeutic Monitoring of Cannabidiol and 7-Hydroxy-cannabidiol in Human Serum and Saliva.

Cannabidiol is a novel antiseizure medication approved in Europe and the US for the treatment of seizures associated with Lennox-Gastaut syndrome, Dravet syndrome and tuberous sclerosis complex. We describe in this article a new and simple liquid chromatography-mass spectrometry method (LC-MS/MS) for the determination of cannabidiol and its active metabolite 7-hydroxy-cannabidiol in microvolumes of serum and saliva (50 μl), to be used as a tool for therapeutic drug monitoring (TDM) and pharmacokinetic studies. After on-line solid phase extraction cannabidiol, 7-hydroxy-cannabidiol and the internal standard cannabidiol-d3 are separated on a monolithic C18 column under gradient conditions. Calibration curves are linear within the validated concentration range (10–1,000 ng/ml for cannabidiol and 5–500 ng/ml for 7-hydroxy-cannabidiol). The method is accurate (intraday and interday accuracy within 94–112% for cannabidiol, 91–109% for 7-hydroxy-cannabidiol), precise (intraday and interday precision <11.6% for cannabidiol and <11.7% for 7- hydroxy-cannabidiol) and sensitive, with a LOQ of 2.5 ng/ml for cannabidiol and 5 ng/ml for 7-hydroxy-cannabidiol. The stability of the analytes was confirmed under different storage conditions. Extraction recoveries were in the range of 81–129% for cannabidiol and 100–113% for 7-hydroxy-cannabidiol. The applicability of the method to TDM was demonstrated by analysis of human serum and saliva samples obtained from patients with epilepsy treated with cannabidiol.

Simultaneous determination of abiraterone and its five metabolites in human plasma by LC-MS/MS: Application to pharmacokinetic study in healthy Chinese subjects

In this study, a rapid, simple and sensitive liquid chromatography–tandem mass spectrometry (LC-MS/MS) method was developed and validated to simultaneously quantify abiraterone (ABI), a widely used anti-metastatic castration-resistant prostate cancer drug, and its metabolites comprising Δ4-abiraterone (D4A), 3-keto-5α-abiraterone (5αA), abiraterone N-oxide (A-NO), abiraterone sulfate (A-Sul) and abiraterone N-oxide sulfate (A-NO-Sul) in human plasma. The analytes were extracted by protein precipitation with acetonitrile and ideal chromatographic separation was achieved on ACE-C18 column (2.1 × 50 mm, 5 µm) using a gradient elution. Triple Quad™ 6500+ mass spectrometer equipped with an electrospray ionization (ESI) source was used and the multiple reaction mode (MRM) was performed. In terms of method validation, good linearity was observed in preassigned validated concentration range for each analyte of interest. Both intra- and inter-batch accuracy was within the range of 87.6–113.8% for all analytes, while intra- and inter-batch precision was below 14.0%. Additionally, both low matrix effects and high recovery were obtained. All analytes remained stable in human plasma at room temperature for 4 h, on wet ice for 8 h, at − 80 °C for 42 d, over three freeze-thaw cycles and under auto-sampler temperature (4 °C) for 48 h post sample preparation. Subsequently, the validated LC-MS/MS method was applied for pharmacokinetic study in healthy Chinese volunteers following an oral dose of 250 mg abiraterone acetate tablet under fasted conditions. Our study for the first time reported the pharmacokinetic parameters of the ABI metabolites in Chinese subjects.

Development and validation of an LC-MS/MS assay for the quantification of cintirorgon (LYC-55716) in mouse plasma and tissue homogenates

Cintirorgon (LYC-55716) is a promising first-in-class antitumor agent as a RORγ agonist in the treatment against various types of cancer. To support preclinical mouse studies, a bioanalytical method was developed and successfully applied for quantification of cintirorgon in mouse plasma and tissue homogenates using LC-MS/MS. The method was fully validated in mouse plasma and partial validation was performed in eight different homogenates originating from brain, kidney, liver, lung, small intestine, small intestine content, spleen, and testis. Sample preparation was performed using 96-well plates for fast and efficient analysis. Protein precipitation was done by addition of 20 µL acetonitrile containing monensin as internal standard to 10 µL sample. Chromatographic separation was achieved on a Polaris 3 C18-A column using gradient elution with 0.2% (v/v) formic acid and 0.2% (v/v) ammonium hydroxide in water (A) and methanol (B) as eluents. The total run time was 3 min. Detection was carried out with a triple quadrupole mass spectrometer with electrospray ionization operated in the positive ion-mode. Quantification could be accomplished within a linear validated concentration range of 5–4,000 ng/mL (10–4,000 ng/mL in brain homogenates) with an intra- and inter-day precision between 4.6–14.7% and 5.1–15.6% (including the LLOQ), respectively, and accuracies between 89.1%–111.2%. The method was successfully applied to a preclinical study with cintirorgon in mice.

Simultaneous Determination of 25 Ginsenosides by UPLC-HRMS via Quantitative Analysis of Multicomponents by Single Marker.

A method using UPLC-HRMS has been developed for a rapid, simultaneous qualitative and quantitative analysis of twenty-five ginsenosides. Chromatographic separation was achieved on a C18 analytical column with an elution gradient comprising 0.1% aqueous formate/acetonitrile as the mobile phase. HRMS detection acquired full mass data for quantification and fullms-ddms2 (i.e., data-dependent scan mode) yielded product ion spectra for identification. Furthermore, quantitative analysis of multiginsenosides by single marker (QAMS) was developed and validated using a relative correction factor. Under optimal conditions, we could simultaneously separate eight groups of isomers of the 25 ginsenosides. Good linearity was observed over the validated concentration range for each analyte (r2 > 0.9924), showing excellent sensitivity (LODs, 0.003–0.349 ng/mL) and lower limit quantification (LOQs, 0.015–1.163 ng/mL). The LC-MS external standard method (ESM) and QAMS were compared and successfully applied to analyze the ginsenoside content from Panax ginseng roots. Overall, our UPLC-HRMS/QAMS approach provides high precision, stability, and reproducibility and can be used for high-throughput analysis of complex ginsenosides and quantitative analysis of multiple components and quality control of traditional Chinese medicines (TCM).

Validated Gas Chromatography – Mass Spectrometry (GC-MS) Method for Simultaneous Quantitation of Tris(4-Chlorophenyl)Methane and Tris(4-Chlorophenyl)Methanol in Rat Plasma and Fetus

Tris(4-chlorophenyl)methane (TCPMe) is a byproduct of dichlorodiphenyltrichloroethane synthesis. TCPMe and its metabolite tris(4-chlorophenyl)methanol (TCPMOH) are environmentally prevalent and have been detected in wildlife and humans. Due to inadequate data addressing its toxicity, the National Toxicology Program (NTP) is testing TCPMe in Hsd:Sprague Dawley SD (HSD) rats following perinatal exposure. In support of the toxicology studies, a gas chromatography – mass spectrometry (GC-MS) method was validated to simultaneously quantitate TCPMe and TCPMOH in male Sprague Dawley rat plasma (primary matrix) over calibration standard ranges of 2–200 ng/mL and 1–100 ng/mL, respectively. The method was linear (r2 ≥ 0.9975), accurate (relative error (RE) ≤ ±12.8 (TCPMe) and ±14.5% (TCPMOH), and precise (relative standard deviation (RSD) ≤ 7.6 (TCPMe) and 3.8% (TCPMOH)). The limits of quantitation were 2 and 1 ng/mL and the limits of detection were 0.73 and 0.07 ng/mL for TCPMe and TCPMOH, respectively. Samples, as high as 2000 ng/mL for TCPMe (RSD ≤ 3.9, RE ≤ ±1.0) and 1000 ng/mL for TCPMOH (RSD ≤ 1.0, RE ≤ ±2.9), were successfully diluted with plasma into the validated concentration range. The method was selective and both TCPMe and TCPMOH were quantified in all secondary matrices (HSD male and female plasma, gestation day (GD)18 plasma, amniotic fluid, and fetus, postnatal day (PND)4 dam and pup plasma) using a primary matrix curve (RE ≤ ±14.4% and 11.5; RSD ≤ 7.6 and 11.8%, respectively). These results demonstrate that the method is suitable for simultaneous quantitation of TCPMe and TCPMOH in rodent plasma and fetuses for the evaluation of gestational and lactational transfer following perinatal exposure of TCPMe in NTP studies.

Development and Validation of an LC-MS/MS Assay for the Quantification of Cintirorgon (LYC-55716) in Mouse Plasma and Tissue Homogenates

Cintirorgon (LYC-55716) is a promising first-in-class antitumor agent as a RORγ agonist in the treatment against various types of cancer. To support preclinical mouse studies, a bioanalytical method was developed and successfully applied for quantification of cintirorgon in mouse plasma and tissue homogenates using LC-MS/MS. The method was fully validated in mouse plasma and partial validation was performed in eight different homogenates originating from brain, kidney, liver, lung, small intestine, small intestine content, spleen, and testis. Sample preparation was performed using 96-well plates for fast and efficient analysis. Protein precipitation was done by addition of 20 µL acetonitrile containing monensin as internal standard to 10 µL sample. Chromatographic separation was achieved on a Polaris 3 C18-A column using gradient elution with 0.2% (v/v) formic acid and 0.2% (v/v) ammonium hydroxide in water (A) and methanol (B) as eluents. The total run time was 3 minutes. Detection was carried out with a triple quadrupole mass spectrometer with electrospray ionization operated in the positive ion-mode. Quantification could be accomplished within a linear validated concentration range of 5-4,000 ng/mL (10-4,000 ng/mL in brain homogenates) with an intra- and inter-day precision between 4.6-14.7% and 5.1-15.6% (including the LLOQ), respectively, and accuracies between 89.1-111.2%. The method was successfully applied to a preclinical study with cintirorgon in mice.

Determination of Perampanel in Dried Plasma Spots: Applicability to Therapeutic Drug Monitoring.

Although therapeutic drug monitoring of antiepileptic drugs is typically based on the analysis of plasma samples, alternative matrices, such as dried plasma spots (DPSs), may offer specific advantages. The aims of this work were to (1) develop and validate a bioanalytical method for the quantitative determination of the second-generation antiepileptic drug perampanel in DPSs; (2) assess short- and long-term stability of perampanel in DPSs; and (3) test the clinical applicability of the developed method. Two hundred microliters of plasma were dispensed on a glass paper filter and dried. Glass paper filter discs were then inserted into clean tubes. After addition of the internal standard (ie, promethazine), the analytes were extracted with 5-mL methanol, dried at room temperature (23 ± 2°C), and reconstituted. Separation and quantification were achieved on 2 serial reverse-phase monolithic columns connected to an UV detector (λ = 320 nm). Calibration curves were linear in the validated concentration range (25-1000 ng/mL). Intraday and interday accuracy were in the range of 99.2%-111.4%, whereas intraday and interday precision (coefficient of variation) ranged from 2.8% to 8.6%. The lowest limit of quantitation was 25 ng/mL. The stability of the analyte in DPSs was assessed and confirmed under different storage conditions. Perampanel concentrations estimated in DPS samples from patients receiving therapeutic doses were equivalent to those measured in plasma samples. This simple method enables the quantitation of perampanel in DPSs with adequate accuracy, precision, specificity, and sensitivity. The short- and long-term stabilities of perampanel in DPSs are highly beneficial for sample shipment or storage at ambient temperature. Moreover, DPSs decreases the costs associated with storage and transportation compared with conventional wet samples.

A high-throughput LC-MS/MS method for determination of flunarizine in human plasma: Pharmacokinetic study with different doses.

A high-throughput and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the determination of flunarizine in human plasma. Liquid-liquid extraction under acidic conditions was used to extract flunarizine and flunarizine-d8 from 100 μL human plasma. The mean extraction recovery obtained for flunarizine was 98.85% without compromising the sensitivity of the method. The chromatographic separation was performed on Hypersil Gold C18 (50 × 2.1 mm, 3 μm) column using methanol-10 mm ammonium formate, pH 3.0 (90:10, v/v) as the mobile phase. A tandem mass spectrometer (API-5500) equipped with an electrospray ionization source in the positive ion mode was used for detection of flunarizine. Multiple reaction monitoring was selected for quantitation using the transitions, m/z 405.2 → 203.2 for flunarizine and m/z 413.1 → 203.2 for flunarizine-d8. The validated concentration range was established from 0.10 to 100 ng/mL. The accuracy (96.1-103.1%), intra-batch and inter-batch precision (CV ≤ 5.2%) were satisfactory and the drug was stable in human plasma under all tested conditions. The method was used to evaluate the pharmacokinetics of 5 and 10 mg flunarizine tablet formulation in 24 healthy subjects. The pharmacokinetic parameters Cmax and AUC were dose-proportional.

Development and validation of a high-performance liquid chromatography-tandem mass spectrometry assay for the quantification of Dexamphetamine in human plasma

Dexamphetamine is registered for the treatment of attention deficit hyperactivity disorder and narcolepsy. Current research has highlighted the possible application of dexamphetamine in the treatment of cocaine addiction. To support clinical pharmacologic trials a new simple, fast, and sensitive assay for the quantification of dexamphetamine in human plasma using liquid chromatography tandem mass spectrometry (LC–MS/MS) was developed. Additionally, it is the first reported LC–MS assay with these advantages to be fully validated according to current US FDA and EMA guidelines.Human plasma samples were collected on an outpatient basis and stored at nominally −20°C.The analyte and the internal standard (stable isotopically labeled dexamphetamine) were extracted using double liquid–liquid extraction (plasma-organic and organic-water) combined with snap-freezing. The aqueous extract was filtered and 2μL was injected on a C18-column with isocratic elution and analyzed with triple quadrupole mass spectrometry in positive ion mode.The validated concentration range was from 2.5–250ng/mL and the calibration model was linear. A weighting factor of 1 over the squared concentration was applied and correlation coefficients of 0.997 or better were obtained. At all concentrations the bias was within ±15% of the nominal concentrations and imprecision was ≤15%. All results were within the acceptance criteria of the latest US FDA guidance and EMA guidelines on method validation.In conclusion, the developed method to quantify dexamphetamine in human plasma was fit to support a clinical study with slow-release dexamphetamine.

Simultaneous quantitation of six aconitum alkaloids and three flavonoids in the herb couple of radix aconiti lateralis-radix glycyrrhizae (Fuzi-Gancao) by UHPLC-ESI-MS/MS.

Background:Fuzi–Gancao herb couple is one of the commonly used herb couples involved in the traditional Chinese medicine formulations, with radix aconiti lateralis (Fuzi in Chinese) and radix glycyrrhizae (Gancao in Chinese) as a ratio of 1:1. Alkaloids and flavonoids were considered as the main active ingredients of Fuzi–Gancao herb couple. However, no analytical methods have been reported to quantitatively analyze these activity ingredients in Fuzi–Gancao herb couple simultaneously.Objective:To develop a simple, rapid, and sensitive UHPLC-MS/MS method for simultaneous quantitation of six alkaloid and three flavonoid compounds, namely, aconitine (AC), mesaconitine (MA), hypaconitine (HA), benzoylaconitine (BAC), benzoylmesaconitine (BMA), benzoylhypaconitine (BHA), liquiritin, isoliquiritin, and licochalcone A (LCA) in Fuzi–Gancao herb couple.Materials and Methods:The chromatographic separation was achieved using a reversed phase C18 column and a mobile phase consisted of water (0.1% formic acid in water, v/v) and acetonitrile. The detection was achieved in multiple reaction monitoring modes. The optimal mass transition ion pairs (m/z) for quantitation were 646.3/586.5 for AC, 632.4/572.5 for MA, 616.3/556.4 for HA, 604.5/572.5 for BAC, 590.4/540.4 for BMA, 574.1/542.5 for BHA, 419.4/257.1 for liquiritin, 419.4/257.1 for isoliquiritin, and 339.2/121.1 for LCA.Results:Good linearity was observed in validated concentration range for each analyte (r > 0.9992), and the intra- and inter-day precisions were <3.12% and 3.65%, respectively. The recovery ranged from 85.36% to 110.14%.Conclusions:The results demonstrated that the method developed was reliable, rapid, and specific. Moreover, this method was successfully applied to control the quality of Fuzi–Gancao herb couple.SUMMARY A simple, rapid, and sensitive UHPLC-MS/MS method for simultaneous quantitation of six alkaloid and three flavonoid compounds in Fuzi–Gancao herb couple has been developed. Abbreviations used: UHPLC-ESI-MS/MS: Ultra High Performance Liquid Chromatography-electrospray ionization-mass spectrometry; RSD: Relative standard deviations; LOD: Limit of detection; LOQ: Limit of quantification; MRM: Multiple reaction monitor; TCMs: Traditional Chinese medicines.

Validation of Ti(III) as a reducing agent in the chemiluminescent determination of nitrate and nitrite in seawater

Titanium (III) trichloride is validated here for the quantitative conversion of all nitrate plus nitrite in seawater to nitric oxide gas, thereby providing an alternative to the typically used reducing agent, ferrous ammonium sulfate plus ammonium molybdate, in the chemiluminescent detection of nitrate plus nitrite at the nanomolar level. We find that both Fe(II)+Mo(VI) and Ti(III) methods yield identical results for standards and seawater samples over a validated concentration range of 1 to 1000nM, and are both in agreement with traditional colorimetric results. Benefits of the Ti(III) reduction chemistry are: simpler preparation, decreased ammonium contamination in a laboratory that measures low-level nutrients, 30% reduction of the sulfuric acid catalyst, and a higher sample through-put. Most importantly, though, this work can be considered the first step on a path toward a much-needed, direct measurement of dissolved organic nitrogen concentrations, as has already been achieved for dissolved organic carbon.

Discrimination and quantification of two isomeric antineoplastic drugs by rapid and non-invasive analytical control using a handheld Raman spectrometer

Raman spectroscopy is a rapid, non-destructive and non-invasive method that is a promising tool for real-time analytical control of drug concentrations. This study evaluated a handheld Raman device to discriminate and quantify two isomeric drugs used to treat cancer. Doxorubicin (DOXO) and epirubicin (EPIR) samples were analyzed at therapeutic concentrations from 0.1 to 2mg/mL (n=90) and 0.08–2mg/mL (n=90) by non-invasive measurements using a portable Raman spectrometer.The discrimination of these two molecules was demonstrated for all concentrations (n=180) by qualitative analysis using partial least square discriminant analysis (PLS-DA) with 100% classification accuracy, sensitivity and specificity and 0% error rate. For each molecule, quantitative analyses were performed using PLS regression. The validity of the model was evaluated using root mean square error of cross validation (RMSECV) and prediction (RMSEP) that furnished 0.05 and 0.02mg/mL for DOXO and 0.17 and 0.16mg/mL for EPIR after pretreatment optimization. Based on the accuracy profile, the linearity range was from 1.256 to 2.000mg/mL for DOXO (R2=0.9988) and from 0.553 to 2.000mg/Ml for EPIR (R2=0.9240) and repeatability (CV% max of 1.8% for DOXO and 3.2% for EPIR) and intermediate precision (CV% max of 2.8% for DOXO and 4.5% for EPIR) were both acceptable.Despite the narrow validated concentration range for quantitative analysis, this study shows the potential of a handheld Raman spectrometer coupled to chemometric approaches for real-time quantification of cytotoxic drugs, as well for discriminating between two drugs with similar UV absorption profiles. Finally, the use of a handheld spectrometer with the possibility of a direct measurement of substances in containers is a potentially valuable tool for combining patient safety with security of healthcare workers.

Determination of tapentadol and tapentadol-O-glucuronide in human serum samples by UPLC-MS/MS.

Tapentadol is a novel, centrally acting analgesic with 2 mechanisms of action, MOR agonism and noradrenaline (NA) reuptake inhibition in a single molecule. It is the first member of a new therapeutic class, MOR-NRI. A high throughput liquid chromatography-tandem mass spectrometric (LC-MS/MS) assay was developed and validated for the quantitative analysis of tapentadol and its O-glucuronide metabolite in human serum. Simultaneous quantification was deemed to be challenging because of the large difference in concentrations between tapentadol and its O-glucuronide metabolite in clinical samples. Therefore, a method was established using a common processed sample, but with different injection volumes and chromatographic conditions for each analyte. Tapentadol and tapentadol-O-glucuronide were determined by protein precipitation of 0.100ml of the samples with acetonitrile. The internal standards used are D₆-tapentadol and D₆-tapentadol-O-glucuronide. The validated concentration range was 0.200-200 ng/ml (tapentadol) and 10.0-10,000 ng/ml (tapentadol-O-glucuronide). Chromatographic separation was achieved by gradient elution on a Waters Acquity UPLC BEH C18 (1.7 μm, 2.1 × 50 mm) column, with mobile phase consisting of 0.01 M ammonium formate (adjusted to pH 4 using formic acid) (A) and methanol (B). A separate injection was done for measurement of each analyte, with a different gradient and run time. The analytes were detected by using an electrospray ion source on a triple quadrupole mass spectrometer operating in positive ionization mode. The run time was 1.6 min for tapentadol and 1.5 min for tapentadol-O-glucuronide. The high sensitivity and acceptable performance of the assay allowed its application to the analysis of serum samples in clinical trials. The validated method was used for analysis of tapentadol in over 17,000 samples.

Determination of Riociguat and its Major Human Metabolite M-1 in Human Plasma by Stable-Isotope Dilution LCMS/MS

Riociguat (BAY 63-2521) is an oral NO-independent as well as NO-synergistic stimulator of soluble guanylate cyclase (sGC) for the treatment of pulmonary hypertension. BAY 60-4552 (M-1) is its pharmacologically active major metabolite. An isotope dilution LC-ESI-MS/MS method has been developed and validated for the simultaneous determination of riociguat and M-1 in lithium heparinized human plasma. The validated concentration range covers 0.500μg/l (LLOQ) to 100μg/l (ULOQ) for both analytes. Interassay accuracy and precision (%CV) for both analytes ranged from 92.7 to 111% and from 2.61 to 9.89%, respectively. The method proved to be selective, specific, sufficiently sensitive, highly reproducible and robust for the analysis of large numbers of samples.

Development of a sensitive LC-MS/MS method for simultaneous quantification of eleven constituents in rat serum and its application to a pharmacokinetic study of a Chinese medicine Shengmai injection.

A sensitive LC-MS/MS method was developed and validated for simultaneous quantification of 11 constituents, ginsenoside Rg1, Re, Rf, Rg2, Rb1, Rd, Rc, ophiopogonin D, schisandrin, schisandrol B and schizandrin B, in rat serum using digoxin as the internal standard (IS). The serum samples were pretreated and extracted with a two-step liquid-liquid extraction. Chromatographic separation was achieved on a C18 analytical column with a proper gradient elution using 0.02% acetic acid aqueous solution and 0.02% acetic acid-acetonitrile as mobile phase at a flow rate of 0.5 mL/min. MS detection was performed using multiple reaction monitoring via an electrospray ionization source. Good linearity was observed in the validated concentration range for every analyte (r(2) ≥0.9929), and the lower limits of quantitation of the analytes were in the range of 0.044-1.190 ng/mL in rat serum. Intra- and inter-day precisions were <14.2%. The accuracy expressed as recovery was within the range of 85.1-112.8%. The extraction recoveries were >75.8%.The validated method was successfully applied to a pharmacokinetic study of all analytes in rats after single intravenous administration of Shengmai injection.

Determination of regorafenib (BAY 73-4506) and its major human metabolites BAY 75-7495 (M-2) and BAY 81-8752 (M-5) in human plasma by stable-isotope dilution liquid chromatography-tandem mass spectrometry.

Regorafenib (BAY 73-4506, commercial name Stivarga(®)) is an oral multikinase inhibitor developed by Bayer Pharma AG (Germany) that targets angiogenic, stromal and oncogenic receptor tyrosine kinases. An isotope dilution liquid chromatography-tandem mass spectrometry method has been developed and validated for the simultaneous determination of regorafenib and its two major metabolites BAY 75-7495 (M-2) and BAY 81-8752 (M-5) in lithium-heparinized human plasma. Analysis was performed after protein precipitation on a triple-quadrupole tandem mass spectrometer. The validated concentration range was from 2.00 (lower limit of quantitation) to 2000 µg/l for all the analytes. Selectivity and specificity in the presence of four potential comedications were demonstrated. Interassay accuracy and precision coefficient of variation ranged from 91.2% (at LLOQ of M-5) to 105% and 3.59 to 14.1%, respectively. The method proved to be selective, specific, sufficiently sensitive, highly reproducible and robust for the analysis of large numbers of samples. The method performance met current guidelines on bioanalytical method validation.

Validation of an in-line Raman spectroscopic method for continuous active pharmaceutical ingredient quantification during pharmaceutical hot-melt extrusion

A calibration model for in-line API determination was developed based on Raman spectra collected during hot-melt extrusion. This predictive model was validated by calculating the accuracy profile based on the analysis results of validation experiments. Furthermore, based on the data of the accuracy profile, the measurement uncertainty was determined. Finally, the robustness of the model was evaluated.A Raman probe was implemented in the die of a twin-screw extruder, to monitor the drug concentration during extrusion of physical mixtures containing 15, 20, 25, 30 and 35% (w/w) metoprolol tartrate (MPT) in Eudragit® RS PO, an amorphous copolymer of acrylic and methacrylic acid esters with a low content of quaternary ammonium groups, which are present as salts. Several different calibration models for the prediction of the MPT content were developed, based on the use of single spectra or averaged spectra, and using partial least squares (PLS) regression or multivariate curve resolution (MCR). These predictive models were validated by extruding and monitoring mixtures containing 17.5, 22.5, 25.0, 27.5 and 32.5% (w/w) MPT. Each validated concentration was monitored on three different days, by two different operators. The β-expectation tolerance intervals were calculated for each model and for each of the validated MPT concentration levels (β was set at 95%), and acceptance limits were set at 10% (relative bias), indicating that at least 95% of future measurements should not deviate more than 10% from the true value. The only model where these acceptance limits were not exceeded was the MCR model based on averaged Raman spectra. The uncertainty measurements for this model showed that the unknown true value can be found at a maximum of ±7.00% around the measured result, with a confidence level of 95%. The robustness of this model was evaluated via an experimental design varying throughput, screw speed and barrel temperature. The robustness designs showed no significant influence of any of the process settings on the predicted concentration values.Raman spectroscopy proved to be a fast, non-destructive and reliable method for the quantification of MPT during hot-melt extrusion. From the accuracy profile of the MCR model based on averaged spectra, it was concluded that for each MPT concentration in the validated concentration range, 95 out 100 future routine measurements will be included within the acceptance limits (10%).