Current Pharmaceutical Analysis - Volume 17, Issue 7, 2021

Background: Antibody Drug Conjugates (ADCs) are complex hybrid molecules comprised of a monoclonal antibody (mAb) connected to a small molecule drug through a linker. The key step in the production of ADCs is bringing together the protein in an aqueous buffer with a hydrophobic small molecule in order to achieve conjugation of the molecules. This step involves dissolving the small molecule portion of the compound in an aqueous miscible organic solvent. These solvents and unconjugated small molecules are ideally cleared by downstream processing in order to achieve the desired product quality. As part of the control system to ensure product quality, the determination of residual solvents in pharmaceuticals is of significant importance in order to protect patient safety and ensure an efficacious drug. Objective: Headspace gas chromatography (HS-GC) is the most widely used tool for quantification of residual solvents for small molecule active pharmaceutical ingredients (APIs) but is not widely used for the analysis of protein-containing samples. In this study, the detection of residual solvents in headspace injections was explored using various conditions in order to detect commonly used conjugation solvents including N,N-dimethylacetamide (DMA), N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), Ethylene Glycol (EG), and Propylene Glycol (PG) in an ADC drug product sample. Methods: Various organic solvents were explored to enhance the response observed with complex protein and residual solvent matrixes. As EG and PG do not partition into the headspace efficiently in the ADC drug product samples that contain large amounts of water, ionic liquids and other ionic compounds were screened with the ADC samples to see if they could improve the partitioning of the key solvents EG and PG. Results: Following headspace and chromatographic optimization, we have developed an approach for the detection and quantification of several conjugation reaction solvents in ADC samples. Conclusion: This new approach is an HS-GC method that simplifies Gas Chromatography (GC) analysis and sample preparation and can be readily implemented in quality control testing for bioconjugated products.

Background: Banlangen granules are broad-spectrum effective antiviral drugs, and have a large clinical demand in China. Free amino acid is one of the main antiviral active ingredients of Banlangen granules. The pre-processing of samples by the existing pre-column derivatization reversed- HPLC method is complicated. Therefore, the determination of free amino acids (AAs) by underivatized ion-pair HPLC-CAD is advantageous for simplifying the preparation process and improving sensitivity. Objective: To better optimize AAs analysis methods, here a sensitive SPE-HPLC-CAD method with a better resolution was established for the determination of underivatized AAs in Banlangen Granule for the first time. Methods: The analytes were separated by HPLC using a Hypercarb column with gradient elution of solvent A (20 mM nonafluorovaleric acid in water) and solvent B (0.3% trifluoroacetic acid in acetonitrile- 0.3% trifluoroacetic acid in water (1:9, v/v)) at a flow rate of 0.15 mL/min. N2 gas pressure and evaporation temperature of CAD were held at a constant 58.6 psi and 60 °C respectively. Results: This method was linear over the respective concentration range of six amino acids. The precision, accuracy, stability and recovery were satisfactory in all samples examined. And the method was successfully applied to the determination of free amino acids in Banlangen granules and its fractions. The total contents of six amino acids in 28 batches of Banlangen Granule were between 1.36 mg/g- 11.62 mg/g. Conclusion: The proposed method could be a simple, accurate and sensitive alternative approach for the determination of free AAs in Banlangen Granule.

Objective: To develop a rapid ultra-performance liquid chromatographic (UHPLC)-UV method for vancomycin determination in human serum for therapeutic drug monitoring (TDM). Methods: Human serum samples were precipitated with 10% perchloric acid, and the supernatant after centrifugation was analyzed on an ACQUITY UHPLC BEH C18 column (2.1 × 50mm, 1.7 μm) via gradient elution with a flow rate at 0.3 mL/min. The mobile phase consisted of acetonitrile and 0.005M KH2PO4 buffer (containing 0.1% triethylamine, pH 3.4). The detection wavelength was set at 210 nm, and the column temperature was set at 40°C. The total runtime was 6.0 min per analysis. Results: After comprehensive validation, the method was applied to determine the concentration of vancomycin in human serum. The chromatographic peaks of vancomycin and internal standard were not interfered by endogenous matrices. The Retention Time (RT) of vancomycin was 1.91 min, while the internal standard was 1.58 min. The good linearity range of vancomycin concentration was 2.5-120 μg/mL (R²>0.999). The lower Limit of Quantitation (LLOQ) was 2.5 μg/mL. The precision at three Quality Control (QC) levels (including LLOQ) was restricted within 85-115%. The extraction recovery rate of QC samples (4.0, 20.0, and 60.0 μg/mL) were 101.16%, 97.70%, and 94.90%, respectively. Inter- and intra-day precision was less than 8% (RSD). Stability tests under different storage conditions were satisfactory. In patients, the concentration of vancomycin ranged from 7.30 to 89.12 μg/mL determined by the fully validated method. Conclusion: The rapid sample pre-treatment procedures and short analysis time made this UHPLC-UV method suitable for therapeutic drug monitoring (TDM) of vancomycin.

Background: Mezlocillin is off-label used for the treatment of respiratory infections in children. Therapeutic Drug Monitoring (TDM) data are also limited in children. A sensitive Liquid chromatography- tandem mass spectrometry (LC–MS/MS) method adapted to children was developed and validated for the determination of mezlocillin plasma concentration in the present study. Methods: Mezlocillin, extracted from a volume of 50 μL plasma using acetonitrile, was analyzed on an online LC-MS/MS system with an Agilent 1290 Infinity UHPLC (Agilent Technologies, CA, USA) coupled to an AB SCIEX QTRAP 6500PLUS MS/MS (AB Sciex, Framingham, MA, USA) with ceftiofur as an internal standard. HPLC separation was performed on a C18 column with ultra-pure water and acetonitrile as gradient elution at a flow rate of 0.4 mL/min at 30°C. Analyst TM Version 1.5.2 (Applied Biosystems) was used for data acquisition. The total chromatographic run time was 1.6 min. Results: LC/MS/MS method used for TDM of mezlocillin in children was developed and validated. This assay has a lower limit of quantification of 0.025 μg/mL for mezlocillin with 50 μL plasma. Good linearity was achieved for mezlocillin over the range from 0.025 to 20 μg /mL. The acceptance criteria were met in all cases. Among 36 patients aged between 0.16-1.63 years old, only one patient had detectable trough concentration higher than 1 μg/mL. Conclusion: LC-MS/MS method with 50 μL plasma developed in this study was successfully applied to TDM of mezlocillin in children. The high variability of trough concentration highlighted that TDM is important to optimize mezlocillin therapy in children.

Introduction: OC26, an ortho-aryl chalcone compound, shows excellent antitumor activity in vitro and vivo. However, the pharmacokinetic characteristics of OC26 have not been comprehensively reported. It is essential to investigate the correlation of pharmacological response. Objective: To further explore OC26, this study aims to develop an ultra-performance liquid chromatography- tandem mass spectrometric (UPLC-MS/MS) method to reveal the pharmacokinetics and distribution characteristics in rats of OC26. Methods: An UPLC-MS/MS method was developed to detect OC26 in plasma and various tissues. The protein precipitation method was applied to process the biological samples. After intravenous injection 12.5mg/kg of OC26 in rats, plasma and tissue samples were collected from rats and the method was applied to investigate pharmacokinetic and distribution characteristics of OC26. Results: Calibration curve samples of OC26 concentration range from 20 to 2000 ng/mL with the goodness of fit (r²> 0.99). The precisions for the method were within 12.3%, while the accuracies for the method were within ±11% (bias). The matrix effect had no influence on the accuracy and precision of the method. After intravenous injection 12.5mg/kg of OC26 in rats, OC26 was rapidly eliminated (t1/2=31.39±7.87min, MRT0→∞=15.03±2.55min) from rat plasma and widely distributed (Vd=4.83±0.96L/kg) in tissues. The highest concentration of OC26 was detected in the brain in which peak content (~8962.78ng/g at 15min) was over 5-fold higher than that of in other tissues, which prompted new potential targets in the brain. Besides, lung and heart also detected quite a high level of OC26. Benefited from quick elimination in the collected tissues and plasma, long-term accumulation was not observed as chronic toxicity might be less. Conclusion: This UPLC-MS/MS method was successfully applied to detect OC26 and provide a theoretical basis for the further study of OC26.

Background: High-Performance Liquid Chromatography (HPLC) method has been used to detect related impurities of perampanel. However, the detection of impurities is incomplete, and the limits of quantification and detection are high. A sensitive, reliable method is badly needed to be developed and applied for impurity detection of perampanel bulk drug. Objective: Methodologies utilising HPLC and Gas Chromatography (GC) were established and validated for quantitative determination of perampanel and its related impurities (a total of 10 impurities including 2 genotoxic impurities). Methods: The separation was achieved on a Dikma Diamonsil C18 column (250 mm 4.6 mm, 5 μm) with the mobile phase of 0.01 mol/L potassium dihydrogen phosphate solution (A) and acetonitrile (B) in gradient elution mode. The compound 2-bromopropane was determined on an Agilent DB-624 column (0.32 mm 30 m, 1.8 μm) by electron capture detector (μ-ECD) with split injection ratio of 1:5 and proper gradient temperature program. Results: Both HPLC and GC methods were established and validated to be sensitive, accurate and robust according to the International Council for Harmonization (ICH) guidelines. The methods developed were linear in the selected concentration range (R²≥0.9944). The average recovery of all impurities was between 92.6% and 103.3%. The possible production mechanism of impurities during the synthesis and degradation processes of perampanel bulk drug was also discussed. Five impurities were analyzed by liquid chromatography–mass spectrometry (LC-MS). Moreover, two of them were simultaneously characterized by LC-MS, IR and NMR. Conclusion: The HPLC and GC methods were developed and optimized, which could be applied for quantitative detection of the impurities, and further stability study of perampanel.

Background: Indobufen is a drug that hinders the aggregation of platelets by reversibly repressing the cyclooxygenase enzyme, further bringing about diminished thromboxane production. During quality control of indobufen tablets, an unknown impurity was detected. Objective: To characterize an unknown impurity in indobufen tablets. Methods: A new method compatible with mass spectrometry detection was set up. A C18 column at 35 °C with a mobile phase consisting of aqueous buffer (including ammonium formate) and methanol (35: 65, v/v) was used at a flow rate of 1.0 mL/min at 228 nm. High-performance liquid chromatography quadrupole time-of-flight mass spectrometry mass spectrometry (HPLC-Q-TOF MS) was used to identify the impurity with the electrospray ionization (ESI) source in the positive ionization mode. Results: The results of HPLC-Q-TOF MS analysis indicated that the protonated molecule ions [M + H]+ of the unknown impurity was at m/z 312. Preparative LC method was put into practice with a Prep- C18 column with a mobile phase consisting of water and methanol (20: 80, v/v) at a flow rate of 20.0 mL/min at 228 nm. The assignment of the 1D and 2D NMR signals was performed for the unknown impurity. In addition, possible formation of the novel impurity was also studied. Conclusion: An unknown impurity in indobufen tablets was characterized. The impurity was assigned as 2-(4-(1-hydroxy-3-oxoisoindolin-2-yl) phenyl) butanoic acid.

Background: Phospholipids are widely used in food and pharmaceutical industry as functional excipients. In spite of the many analytical methods reported, there are very limited reports concerning systematic research and comparison of phospholipid excipients. Objective: To present a comprehensive evaluation of commercial natural phospholipid excipients (CNPEs). Methods: Seventeen batches of CNPEs from five manufacturing enterprises, isolated either from soybean or egg yolk, were investigated. The content and composition of phospholipids, fatty acids and sterols as a whole were considered as the evaluative index of CNPEs. Eight kinds of phospholipids were determined by Supercritical Fluid Chromatography (SFC), twenty-one kinds of fatty acids were determined by Gas Chromatography (GC) after boron trifluoride-methanol derivatization, and nine kinds of sterols were determined by High Performance Liquid Chromatography (HPLC) after separation and derivatization of the unsaponifiable matter. Cluster analysis was employed for the classification and identification of the CNPEs. Results: The results showed that each kind of CNPEs had its characteristic content and composition of phospholipids, fatty acids and sterols. Seventeen batches of samples were divided into eight groups in cluster analysis. CNPEs of the same type from different source (soybean or egg yolk) or enterprises presented different content and composition of phospholipids, fatty acids and sterols. Conclusion: Each type of CNPEs had its characteristic content and composition of phospholipid, fatty acid and sterol. The compositions of phospholipid, fatty acid and sterol as a whole can be applied as an indicator of the quality and characteristics for CNPEs.

Introduction: Diquat is a fast-acting contact herbicide and plant dehydrating agent. The oral lethal dose 50 (LD50) of diquat in mice is about 125 mg/kg. The purpose of this study is to research the metabolomics in mouse plasma after acute diquat poisoning. Methods: The mice were divided into two groups (the control group and acute diquat poisoning group). The control group was given normal saline by gavage. The acute diquat poisoning group was given 50 mg/kg diquat. UPLC-MS/MS was used to determinate the small molecule organic acid in mouse plasma. Results: Compared to the control group, the L-lysine, Adenine, L-Alanine, L-Valine, Lactic acid, Inosine, Adenosine, L-Tryptophan, L-Tyrosine, L-Arginine, L-Phenylalanine, L-Methionine, Citric acid, Fructose, L-Glutamine, Malic acid, L-Aspartic acid and Pyruvic acid increased in the acute diquat poisoning group (p<0.05); while the L-Histidine decreased (p<0.05). Conclusion: The results of metabolites increased or decreased, indicating that acute diquat poisoning induced amino acid metabolism and energy metabolism perturbations in mice.

Objective: To systematically characterize the impurity profile in Daptomycin raw material by 2 Dimensional LC/MSn. Methods: The target impurity was separated by first Dimensional HPLC and enriched by a 500μl loop, then desalted using the on-line second Dimensional HPLC and analyzed by MS detector in positive mode. Their structures were characterized based on the degradation mechanism and mass fragmentation regularity of the cyclic lipopeptide, as well as the molecular thermodynamic calculation. Results: A total of 12 impurities were characterized in the raw material, including 6 degradation products; 8 impurities are reported for the first time. The mass fragmentation regularities of 2 β-isomers of Asp residue were summarized. Conclusion: The structures of impurities in Daptomycin raw material, especially for β-isomer impurities, could be rapidly identified by on-line 2 Dimensional LC/MSn method together with the molecular thermodynamic calculation.

Background: The number of validated quantification methods for rifampicin, a prototypical Oatp inhibitor, in biological rat samples is limited. Objective: This study was conducted to validate a modified reversed-phase liquid chromatographic method for the determination of rifampicin in rat liver tissue according to the current ICH M10 Bioanalytical Method Validation Draft Guideline (2019) for application to samples of in situ rat liver perfusion studies. Methods: Liver tissue samples were obtained from recirculatory in situ rat liver perfusion studies. The analysis was performed on a C18 column with a mobile phase composed of 0.05 M phosphate buffer (pH 4.58): acetonitrile (55:45, v/v). The assay was validated for selectivity, calibration curve and range, matrix effect, carry-over, accuracy and precision, reinjection reproducibility, and stability. Results: The method was considered selective and stable, without having carry-over and matrix effects. The calibration curve was linear (R²: 0.9983) within the calibration range (0.5-60 ppm). Accuracy and precision values fulfilled the required limits. Liver concentrations of rifampicin in liver tissue, obtained after 60 min perfusion with 10 μM and 50 μM of rifampicin, were 45.1 ± 11.2 and 313.4 ± 84.4 μM, respectively. Conclusion: The bioanalytical method validation was completed and the method was successfully applied for the determination of rifampicin in rat liver tissue.

Background: Buprenorphine is quite common in the illicit market. Buprenorphinecontaining drug abuse is frequently encountered in patients. The analysis methods used to determine the abuse of buprenorphine and norbuprenorphine are important for forensic science. Buprenorphine is metabolized to norbuprenorphine by the liver. Objective: Therefore, the determination of buprenorphine and norbuprenorphine in urine is one of the methods to determine the abuse of buprenorphine. Methods: In this study, we developed a precise, simple, and rapid ultra-performance liquid chromatography- tandem mass spectrometer method for the determination of buprenorphine and norbuprenorphine simultaneously. Results: The developed method was validated in terms of selectivity and linearity, which was in the range of 9–1800 ng/mL for both buprenorphine and norbuprenorphine. The intra-assay and inter-assay accuracy and precision were found within acceptable limits of the EMA guideline. Lower limits of quantitation were 9 ng/mL for both buprenorphine and norbuprenorphine. Conclusion: The developed method was successfully applied for the determination of both analytes in the proficiency testing samples.

Background: In view of the current FDA standardization and product quality control criteria, Quality by design approach for analytical methods is gaining importance to develop a robust analytical method. A new Quality by Design approach by RP-HPLC was developed and validated for the quantification and purification of Tadalafil hydrochloride and its tablet formulations. Objective: The objective of the study was to develop and validate a simple, robust, and accurate method by QbD approach for the detection of Tadalafil hydrochloride and its degradation products in bulk drug and tablet formulation. Methods: The chromatographic separation was performed on JASCO Crest Pack RPC18 column (250mm×4.6mm, 5μm) with mobile phase A consisting of a mixture of Acetonitrile: Methanol (40:20 v/v); and mobile phase B: 0.01M Ammonium acetate in water pH adjusted to 3.50± 0.05 with glacial acetic acid with 1.0ml/min flow rate at 285nm. Box-Behnken's three-level, 3-factorial design was employed to create and analyze a "Design Space" (DoE). This design was statistically analyzed by ANOVA, counter-plot, and 3D response surfaces plots, which demonstrated that the model was statistically significant. The developed method was validated as per the ICH guidelines Q2 (R1). Results and Discussion: The tadalafil hydrochloride showed good regression (R2>0.9995) within tested ranges, and the percent recovery was found to be 98% in the marketed formulation. Conclusion: The method was found to be highly specific without the interference of impurities and degradation products of tadalafil hydrochloride. For quantification and routine quality control of tadalafil and its marketed formulation, the stability-indicating the RP-HPLC method could, thus, be extended.

Background: Diazepam belongs to the group of 1,4-benzodiapines. It is used for the treatment of anxiety, convulsions and as muscle relaxants. The presence of a 4,5-azomethine group enables its electrochemical detection. Introduction: A screen-printed electrode modified with antimony film was used for the determination of diazepam in pharmaceutical preparations. Methods: Electrode modification was done by ex-situ deposition of antimony on a commercially available screen-printed electrode. Parameters affecting the electroanalytical response of the sensor, such as deposition potential, deposition time, and antimony concentration, were examined and optimized. The modified electrode showed enhanced electroactivity for diazepam reduction compared to the unmodified electrode. Under optimal conditions, linear sweep voltammetry was used for the determination of the analyte. Results: The sensor showed linear dependence in the range from 0.5 to 10 μmol/L, the correlation coefficient was 0.9992. The limit of detection was 0.33 μmol/L, the corresponding limit of quantification was 1.08 μmol/L. The modification enabled the determination of diazepam in the presence of oxygen. Conclusion: The modified electrode was used for the determination of diazepam in tablets. Results confirmed the applicability of the electrochemical sensor.