Elution Research Articles

MOF@N–doped carbon dots–based dispersive solid phase extraction combined dispersive liquid–liquid microextraction of several aflatoxins from raw cow’s milk samples prior to HPLC–FLD

A dispersive solid-phase extraction method based on metal–organic framework and nitrogen–doped carbon dot nanocomposite combined with magnetic ionic liquid-based dispersive liquid–liquid microextraction was established for the extraction of aflatoxins from milk samples. For this purpose, the proteins in the sample were precipitated using a zinc sulfate solution. After vortexing (for 5 min) and centrifuging, 35 mg of the sorbent were added into the aqueous phase obtained from previous step, and the mixture was vortexed for five minutes. In the following, the sorbent particles were separated and eluted with methanol in the presence of vortex agitation (4 min). After centrifuging, the eluent phase was mixed with magnetic ionic liquid (1- octyl-3-methylimidazolium tetrachloroferrate) and rapidly injected into deionized water to perform the dispersive liquid–liquid microextraction procedure. Finally, the magnetic solvent was separated in the presence of a magnet and injected into a high-performance liquid chromatography-fluorescence detector after diluting with the mobile phase. Referring to the results, the limits of detection and quantification of the studied aflatoxins were in the ranges of 0.69–1.09 and 2.31–3.63 ng/L, respectively. Extraction recoveries were in the range of 61 to 73 %. At the end, the suggested approach was employed in the analysis of the aflatoxins in the raw cow’s milk samples collected from different regions of East Azarbaijan and AFM1 was found in several samples (34 out of 60 samples). Also, AFB1 was found in three samples.

Poly[1-vinyl-3-benzyltriethylammonium proline ionic liquid] membrane: Preparation, properties and adsorption applications

A novel poly[1-vinyl-3-benzyltriethylammonium proline ionic liquid] membrane(PIL/PVDF membrane) were prepared through the blending poly[1-vinyl-3-benzyltriethylammonium proline ionic liquid](P[Pro][TEA])-poly(vinylidene fluoride)(PVDF) phase inversion method. A series of PIL/PVDF membrane characterization (NMR, FT-IR, SEM, EDS, and XPS) was performed. The properties of PIL/PVDF membrane (contact angle, water flux, mechanical properties, thermal stability,electric charge property, specific surface area and pore diameter) were evaluated. A solid phase extraction − high performance liquid chromatography (SPE-HPLC) method for separation/analysis of quercetin was developed. The factors affecting the adsorption and elution (time, dosage, pH etc.) were investigated. Under optimal conditions, the method has been successfully applied to the separation/analysis of quercetin in tea, and the recovery rates are satisfactory (86.4–106.0 %). The concentration factor of quercetin was 10. The adsorption capacity of PIL/PVDF membrane towards quercetin was 22.1 mg g−1. The extraction mechanism was preliminarily discussed. The established SPE-HPLC method was simple and reliable, which provided application prospects for extraction and detection of quercetin.

A dilute-and-inject liquid-gas chromatography-mass spectrometry method for the analysis of sixteen polycyclic aromatic hydrocarbons in extra-virgin olive oil

BackgroundPolycyclic aromatic hydrocarbons (PAHs) represent a diverse group of organic compounds characterized by the fusion of two or more benzene rings arranged in various structural forms. Due to their harmful effects on human health, it is essential to implement monitoring systems and preventive measures to regulate human exposure. Given the affinity of PAHs for lipids, extensive research has been focused on their presence in vegetable oils. This study aimed to develop an on-line liquid-gas chromatography (LC-GC) method (using tandem mass spectrometry) with minimized solvent consumption for the determination of 16 PAHs in extra-virgin olive oil (EVOO). ResultsA side-by-side comparison of the selected-ion-monitoring and the pseudo multiple-reaction-monitoring (p-MRM) acquisition modes was performed, in terms of specificity and detectability. The results obtained using the p-MRM mode were superior, and for this reason it was selected. The method was linear over the concentration range 1–200 μg kg−1 (except in five cases, over 2–200 and 5–200 μg kg−1 ranges). Accuracy (at the 2 μg kg−1 and 20 μg kg−1 concentration levels) was in the 86.9–109.3 % range, with an RSD <10 %. Intra-day and inter-day precision (at 2 μg kg−1 and 20 μg kg−1 concentration levels) were in the 1.2–9.7 % and 3.2–10.8 % ranges, respectively. For all the PAHs, a negative matrix effect was observed. Three out of sixteen PAHs were detected in three EVOOs (among ten samples), albeit at the low ppb level. Limits of quantification were satisfactory in relation to EU legislation on the presence of PAHs in vegetable oils. SignificanceA dilute-and-inject LC-GC-tandem mass spectrometry method is herein proposed fulfilling EU legislation requirements; sample preparation was very simple, inasmuch that it involved only a dilution step, thus avoiding extraction, clean-up, and thus a high consumption of organic solvents. In fact, considering both oil dilution and the LC mobile phase, less than 8 mL of solvents were used.

Improving the Current European Pharmacopoeia Enantio-Selective HPLC Method for the Determination of Enantiomeric Purity in Atorvastatin Calcium Salt Drug Substance

Atorvastatin (ATV) is a well-established lipid-lowering agent. ATV has two stereogenic centers, and of the four possible stereoisomers, only the (3R,5R) form is used therapeutically. The European Pharmacopoeia (EP) monograph 2022 for ATV calcium salt describes a normal-phase high-performance liquid chromatography (HPLC) method for the determination of enantiomeric purity in both drug substance and working standard samples, based on a 150 mm × 4.6 mm Chiralpak AD-H column. The main problems with this method are the very long analysis time and the high solvent consumption. Here, an alternative chromatographic protocol was developed using the Chiralpak AD-3 column (250 mm × 4.6 mm) packed with 3 μm silica particles instead of the 5 μm silica particles of the Chiralpak AD-H chiral stationary phase and characterized by the same polysaccharide selector, amylose-tris(3,5-dimethylphenylcarbamate). Using a mobile phase consisting of a mixture of n-hexane-ethanol-formic acid 90:10:0.1 (v/v/v) as the mobile phase and setting the flow rate and column temperature to 1.0 mL min−1 and 35 °C, respectively, a simultaneous stereo-selective separation was achieved within 35 min without observing any overlap between the enantiomeric impurity peak and peaks related to other ATV impurities. Compared to HPLC EP conditions, the analysis time to elute all the potentially related substances was faster and significantly less mobile phase volume was required. The linearity of the method has been demonstrated in the range of 4.4 μg mL−1 to 1000 μg mL−1 (R2 &gt; 0.999). At a concentration of 4.4 μg mL−1, which is 0.075% of the test solution (5.8 mg mL−1, as ATV free acid), the signal-to-noise ratio was found to be 20.

A rapid and highly sensitive HILIC-HPLC based analytical assay method for quantification of azacitidine and venetoclax in a single run

Venetoclax and Azacitidine combination has recently been approved by the USFDA to treat old people with acute myeloid leukemia. Pharmaceutical formulations of the combination of these two drugs are expected to be launched soon. Till date, an analytical method of any type has not been reported in literature for quantification of Venetoclax and Azacitidine in a single run. The current work set out to establish an HILIC-HPLC based analytical assay method for simultaneous quantification of Azacitidine and Venetoclax from the mixed sample. The method has been developed employing Poroshell HILIC column. Ammonium formate (10 mM, pH 5.0) and acetonitrile were used as a mobile phase in gradient programme. A variety of parameters, including linearity, accuracy, precision, specificity, robustness, and ruggedness were evaluated to validate the method. Azacitidine and Venetoclax were eluted at 8.8 min and 4.0 min, respectively. Method was linear over the range of 1-100 µg/mL. Recovery was found to be 98.29-99.28% and 101.84-101.95% for Azacitidine and Venetoclax, respectively. The method was reproducible with a relative standard deviation of less than two in precision studies. The developed method can suitably be employed for quantifying Venetoclax and Azacitidine in a single run from their mixed samples.

Development and validation of stability indicating ultra‐high‐performance liquid chromatographic method for assay and impurities of mexiletine hydrochloride and mexiletine hydrochloride capsules

AbstractMexiletine (MEX) is an antiarrhythmic drug used to treat acute and chronic ventricular arrhythmias. A simple isocratic, rapid, specific, stability‐indicating, and sensitive reverse‐phase ultra‐high‐performance liquid chromatography (UHPLC) method was developed and successfully validated to quantify the assay and impurities of MEX hydrochloride. The new UHPLC method showed the optimum separation of MEX and its seven impurities in 20 min by using sodium acetate buffer (0.14 mM) with 0.3% of glacial acetic acid (pH 4.8) and methanol in the ratio of 40:60 (v/v) as mobile phase. The method used an Acquity HSS T3 (100 × 3.0 mm, 1.8 µm) chromatographic column at a 0.2 mL/min flow rate. The injection volume is 2 µL, and 254 nm is the detection wavelength. Note that, 25°C is the column oven temperature. The method is linear over the concentration range of the quantification limit (0.03%) to 150% (0.3%) of the specification level (0.2%) for the impurities and 50%–150% for the assay method. The quality control lab of bulk drugs and finished dosage form successfully applied the new UHPLC method for quantification of assay and impurities of MEX hydrochloride.

DEVELOPMENT AND VALIDATION OF HPLC-METHOD FOR QUANTITATIVE DETERMINATION OF PROPOXUR IN COLLARS AND SPRAYS

Propoxur is a carbamate non-systemic insecticide, an acetylcholinesterase blocker of invertebrates, leading to their paralysis and death. Propoxur is used in insect acaricidal sprays and collars to kill fleas and ticks on pets. The aim of the work was to develop a method of identification and quantification of propoxur in spray for external use and collars with different matrices. The method was developed and validated by indicators of selectivity, linearity, suitability parameters of the chromatographic system and repeatability. Spray and collars with different polymer composition were used as a sample-object for the development of the technique. The standard sample and the test sample of the spray were dissolved in acetonitrile to a propoxur concentration of 50 μg/ml. The collar was dissolved in tetrahydrofuran and acetonitrile was added to precipitate the matrix. The samples were separated on a Dionex Ultimate 3000 chromatograph equipped with chromatographic column Acclaim C18 150×4.6, 5 μm. The mobile phase was a mixture of acetonitrile and water in a volume ratio of 80:20. Propoxur was detected at an absorption wavelength of 270 nm. Under the above conditions, it was possible to completely separate propoxur (retention time of the chromatographic peak – 2.2 min) and other components of the studied drugs. At the same time, the suitability parameters of the chromatographic system did not exceed the limits specified in the recommendations of the USA Food and Drug Association. For propoxur, the efficiency of the chromatographic system was 10,300–10,540 theoretical plates. The relative standard deviation (RSD) for the peak areas of the active substance was ± 0.37%, and the peak separation ratio (RS) of propoxur and other components of the drug was 3.1–5.7. The symmetry coefficient of the propoxur peak was 1.49. The calibration curve was linear in the recommended DFU 2.0 range (80–120% of the nominal concentration of the active substance). The coefficient of linearity (R2) for propoxur was 0.9985.

Quantitative analysis of residual butylated hydroxytoluene and butylated hydroxyanisole in Salmo salar, milk, and butter by liquid chromatography–tandem mass spectrometry

Butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA) are two commonly used antioxidants with potential health risks associated with excessive intake from multiple sources. Several countries have implemented strict regulations to curb these risks. This study presents a simple LC–MS/MS method for estimating BHT and BHA levels in Salmo salar, butter, and milk. To mitigate any potential interference from the three complex matrices with the ionisation of the target analytes, the method utilised the standard addition approach. The mobile phase used to elute the analytes consisted of 0.1 % formic acid in a mixture of water and acetonitrile (25:75 v/v). Both antioxidants were detected in negative ionisation mode. BHT was identified through single-ion monitoring at a mass-to-charge ratio (m/z) of 219.4, while BHA was detected using multiple-reaction monitoring, with a transition from m/z 164.0 to 149.0. The environmental assessment of the applied procedures verified that the approach is eco-friendly.

Emerging mycotoxin occurrence in chicken feed and eggs from Algeria.

Poultry farming has developed into one of Algeria's most productive industrial farming because of the growing demand for sources of protein among Algerian society. Laying hen feed consists mainly of cereals, which can be contaminated with molds and subsequently with their secondary metabolites known as mycotoxins. These later can pose a serious danger to the production and quality of eggs in the commercial layer industry. This work focuses on the detection of emerging mycotoxins, mainly enniatins (ENNs) and beauvericin (BEA), in poultry feed and eggs from different locations in Algeria. Two different QuEChERS-based extractions were established to extract ENNs and BEA from chicken feed and eggs. The determination of mycotoxin occurrence was achieved by a UHPLC-MS/MS method using 0.1% (v/v) formic acid in water and MeOH as mobile phase, an ESI interface operating in positive mode, and a triple quadrupole mass spectrometer operating in MRM for the detection. Matrix-matched calibration curves were carried out for both matrices, obtaining good linearity (R2 > 0.99). The method performance was assessed in terms of extraction recovery (from 87 to 107%), matrix effect (from - 47 to - 86%), precision (RSD < 15%), and limits of quantitation (≤ 1.1µg/kg for feed and ≤ 0.8µg/kg for eggs). The analysis of 10 chicken feed samples and 35 egg samples composed of a 10-egg pool each showed that ENN B1 was the most common mycotoxin (i.e., found in 9 feed samples) with contamination levels ranging from 3.6 to 41.5µg/kg, while BEA was detected only in one feed sample (12µg/kg). However, eggs were not found to be contaminated with any mycotoxin at the detection limit levels. Our findings indicate that the searched mycotoxins are present in traces in feed and absent in eggs. This can be explained by the application of a mycotoxin binder. However, this does not put a stop on the conduction of additional research and ultimately setting regulations to prevent the occurrence of emerging mycotoxins.

Validation and application of an online extraction and liquid chromatography tandem mass spectrometry assay for the analysis of delamanid and its DM-6705 metabolite in human breast milk

We developed and validated a bioanalytical assay to quantify delamanid and its key metabolite (DM-6705) in breast milk and aimed to quantify the secretion of these compounds in breast milk. Due to the hydrophobic nature of the analytes, special care was taken during sample preparation to prevent the formation of fatty deposits during protein precipitation. This was followed by online solid phase extraction and liquid chromatography with tandem mass spectrometry for detection. A Restek Viva BiPh C18 column (1.0 mm×50 mm, 5 µm) was used for extraction while chromatographic separation was performed using a Waters Xterra MS C18 (2.1 mm×100 mm, 5 μm) analytical column with an isocratic mobile phase consisting of acetonitrile, methanol, and 5 mM ammonium carbonate. The mass spectrometric detection of the analytes was performed using an AB Sciex 3200 mass spectrometer employing electrospray ionisation in the positive mode with multiple reaction motoring of the relevant precursor and product ions. Delamanid-d4 and OPC-14714 were used as internal standards. A quadratic (weighted 1/x concentration) regression was used to fit calibration curves for delamanid and DM-6705 over the concentration range of 10.0 – 1000 ng/mL. The intra- and inter-day validation accuracies of the quality control samples were between 92.1% and 98.3% for delamanid, and 97.0% and 102.8% for DM-6705. The percentage coefficient of variation (precision) was less than 7.8%. To our knowledge, this is the first report describing the concentrations of delamanid and DM-6705 in the breast milk of patients treated for rifampicin-resistant tuberculosis.

A Simple, Sensitive, and Greener HPLC-DAD Method for the Simultaneous Analysis of Two Novel Orexin Receptor Antagonists.

The orexin receptor antagonist (ORA) is one of the new psychopharmacological agents used in the treatment of insomnia. There are currently no documented greener high-performance liquid chromatography-diode array detector (HPLC-DAD) methods for the analysis of ORA antagonists, lemborexant (LMB) and suvorexant (SUV) simultaneously. Therefore, in this study, a simple, sensitive, and greener HPLC-DAD method has been developed for the simultaneous quantitative analysis of LMB and SUV in bulk and laboratory-prepared mixture. The developed method was validated for numerous validation parameters and evaluated for greenness. The C18 Waters Spherisorb CN (4.6 × 250 mm2; 5 μm) column was used for the chromatographic separation. The mobile phase composition was ethanol: 10 mM KH2PO4 buffer in a ratio of (60:40 v/v). The DAD detection was performed at 253 nm using a Waters DAD detector. The greenness was evaluated using the analytical Eco-Scale (AES), ChlorTox, and analytical GREEnness (AGREE) techniques. The calibration curves showed excellent linearity for LMB and SUV between the concentration range of 125-5000 ng/mL and 250-10,000 ng/mL, respectively. In addition, the proposed HPLC-DAD method was accurate, precise, robust, highly sensitive, and greener. AES, ChlorTox, and AGREE scales were predicted by the HPLC-DAD method to be 91, 1.14 g, and 0.79, respectively, showing an excellent greenness profile. The greener HPLC-DAD method was successfully used to analyze both medicines quantitatively in bulk and laboratory-prepared synthetic mixtures. The findings of this study indicated that the proposed HPLC-DAD method may be consistently applied to evaluate LMB and SUV in bulk and dosage forms.

Sustainable cannabinoids purification through twin-column recycling chromatography and green solvents

In the present study, twin-column recycling chromatography has been employed for the purification of a Cannabis extract by using a green solvent, ethanol, as the mobile phase. In particular, the complete removal of the psychoactive tetrahydrocannabinol (THC) from a Cannabis extract rich in cannabidiol (CBD) was achieved under continuous conditions. The performance of the method, in terms of compound purity, recovery, productivity and solvent consumption, was compared to that of traditional batch operations showing the potential of the twin-column recycling approach. The employment of a theoretical model to predict the band profiles of the two compounds during the recycling process has facilitated method development, thus further contributing to process sustainability by avoiding trial and error attempts or at least decreasing the number of steps significantly.Graphical abstract

Stability Indicating RP-HPLC Method Development and Validation for Simultaneous Estimation of Metformin Hydrochloride and Remogliflozin Etabonate in Bulk and Tablet Formulation

A simple efficient stability-indicating validated RP-HPLC method has been developed for the simultaneous determination of Metformin hydrochloride (MET) and Remogliflozin etabonate (REM) in bulk and was applied to marketed formulations. The mobile phase used for detection was Phosphate Buffer (pH 4.0): Acetonitrile (60:40%v/v). Drug peaks were detected by a UV detector at 226nm. The linearity of the developed method was found to be 20-60µg/mL for Metformin hydrochloride and 4-12μg/mL for Remogliflozin etabonate. The Limit of Detection (LOD.) and Limit of Quantitation (LOQ.) were found to be 0.085µg/mL and 0.258µg/mL for MET and 0.010µg/mL and 0.030µg/mL for REM respectively. Hydrolysis by HCl, NaOH, hydrogen peroxide, UV light and temperature were performed on a formulation which proves that the proposed method was specific and will be suitable for routine quality control analysis.

Comparing the Greenness and Validation Metrics of Traditional and Eco-Friendly Stability-Indicating HPTLC Methods for Ertugliflozin Determination.

The literature does not provide any "high-performance thin-layer chromatographic (HPTLC)" techniques for the determination of a novel antidiabetic medicine, ertugliflozin (ERZ). Additionally, there are not many environmentally friendly analytical methods for ERZ measurement in the literature. A rapid, sensitive, and eco-friendly reversed-phase-HPTLC (RP-HPTLC) method was designed and validated in an attempt to analyze ERZ in marketed pharmaceutical tablets more precisely, accurately, and sustainably over the traditional normal-phase HPTLC (NP-HPTLC) method. The stationary phases used in the NP- and RP-HPTLC procedures were silica gel 60 NP-18F254S and 60 RP-18F254S plates, respectively. For NP-HPTLC, a chloroform/methanol (85:15 v/v) mobile phase was used. However, ethanol-water (80:20 v/v) was the preferred method for RP-HPTLC. Four distinct methodologies, including the National Environmental Method Index (NEMI), Analytical Eco-Scale (AES), ChlorTox, and Analytical GREEnness (AGREE) approaches, were used to evaluate the greenness of both procedures. For both approaches, ERZ detection was carried out at 199 nm. Using the NP- and RP-HPTLC techniques, the ERZ measurement was linear in the 50-600 and 25-1200 ng/band ranges. The RP-HPTLC method was found to be more robust, accurate, precise, linear, sensitive, and eco-friendly compared to the NP-HPTLC approach. The results of four greenness tools demonstrated that the RP strategy was greener than the NP strategy and all other reported HPLC techniques. The fact that both techniques can assess ERZ when its degradation products are present implies that they both have characteristics that point to stability-indicating features. 87.41 and 99.28%, respectively, were the assay results for ERZ in commercial tablets when utilizing the NP and RP procedures. Based on several validation and greenness metrics, it was determined that the RP-HPTLC approach was better than the NP-HPTLC method. As a result, it is possible to determine ERZ in pharmaceutical products using the RP-HPTLC approach.

Development and Validation of Stability Indicting HPLC Method for the Separation and Simultaneous Analysis of Timolol, Dorzolamide and Latanoprost Inophthalmic formulations

The present work is intended to establish a simple, precise and sensitive stability indicating HPLC method for the separation and simultaneous quantification of timolol, dorzolamide and latanoprost in pharmaceutical formulations. The separation of analytes was achieved on Spherisorb ods2 C18 (250mm × 4.6mm; 5µ)as stationary phase, methanol, acetonitrile and phosphate buffer (pH 5.2) in 55:45:05 (v/v) as mobile phase at 1.0 mL/min and UV detection at 239nm. In this condition, well resolved, retained peaks were identified at 3.45 min fortimolol, 2.66min for dorzolamideand 5.43min for latanoprost. The method reports 0.313µg/mL, 1.25µg/mL and 0.003µg/mL for timolol, dorzolamide and latanoprost respectivelyas LOD that proves that the method have enough sensitivity levels for the detectionanalytes in samples. The method passes all the validation parameters as per the guidelines proved that the method was valid. The method can shows very less % degradation in various stress studies such as acidic, base, peroxide, thermal and UV light conditions and can effectively separate various stress degradation compounds and confirms the stability indicating nature of the method. The method applicability was assessed by analysing the drug content in ophthalmic drops and reports the % assay of be 98.48, 99.37 and 98.32% for timolol, dorzolamide and latanoprost respectively. Based on the results, it can be concluded that the method can adequately suitable for the separation and quantification of timolol, dorzolamide and latanoprost and hence can be applicable for the routine analysis of timolol, dorzolamide and latanoprostin single or any combined ophthalmic formulations.

Analytical Quality by Design as applied to the development of a SEC-HPLC platform procedure for the determination of monoclonal antibody purity without mobile phase additives

This work presents the application of AQbD principles to the development of a size exclusion chromatography (SEC) HPLC procedure for the determination of monoclonal antibody (mAb) product purity using state-of-the-art column technology available via the Waters™ XBridge Premier Protein SEC column. Analytical Quality by Design (AQbD) emphasizes a systematic, risk-based lifecycle approach to analytical procedure development based on sound statistical methodologies. It has recently become increasingly recommended by regulatory agencies as a response to the need for greater efficiency, improved reliability, and increased robustness among modern analytical procedures in the pharmaceutical industry. Use of an Analytical Target Profile (ATP) and formal risk assessments informed the application of Design of Experiments (DoE) to optimize this analytical procedure, as well as assess its robustness and ruggedness. Importantly, our ruggedness results demonstrated the transferability of this procedure between two laboratories within the Catalent Biologics Global Network. Application of this analytical procedure as a platform approach for evaluating mAb purity is expected to support expedited, first-in-human timelines of mAb molecules by enabling great quantitative performance with simple mobile phase buffer compositions. Taken together, this case study demonstrates the utility of adopting AQbD principles in analytical procedure development.

Bioanalytical Method Development and Validation and forced degradation of Sitagliptin and determination of Pharmacokinetic application study in Human Plasma by RP-HPLC method

In the present investigation, an attempt was made for the development, validation, forced degradation and pharmacokinetic application of sitagliptin in the human plasma spiking studies by UV - HPLC method. The experimentation was developed based on the extensive literature survey and ascertained by the statistical parameters of the sampling. A simplified, accurate method was created by the liquid chromatographic system from Shimadazu LC 20AD consisting of manual injection. The optimized chromatogram was obtained with acetonitrile in the isocratic mobile phase method at a 1.0 mL/min flow rate. A thermo C-8 column (4.6X250mm,5μm) was used as a stationary phase, and 265.0nm was selected as the detection wavelength with the aid of a UV-Vis detector. The proposed method was validated as per ICH guidelines. The technique was linear in the range of 10-50μg/mL with correlation coefficient R2 = 0.9746, respectively. Recovery studies postulated that % RSD 19.14, 3 &amp; 9.95 respectively. Injection repeatability values were found to be % RSD 17 &amp; 10.63 for intraday and interday, respectively. Stress degradation studies revealed that sitagliptin degrades more rapidly when subjected to 0.1 NaoH. Human plasma spiking studies reported 3.02 ng/mL at 3.02+/-60 min of C and T max, respectively. Keywords: Sitagliptin, Method development, HPLC, Validation, Stress degradation studies, Human plasma spiking studies

STABILITY BASED HPLC METHOD FOR CYCLOPHOSPHAMIDE RELATED SUBSTANCES IN FINISHED DRUG PRODUCTS: DEVELOPMENT AND VALIDATION

Objective: The current study aimed to develop a simple, sensitive, and precise high-performance liquid chromatographic (HPLC) method for estimating cyclophosphamide and its related substances, as well as to implement the developed method in a capsule product. Methods: Method development was performed using various solvent and buffer-solvent ratios at different flow rates for better resolution and to decrease the run time. The developed method was validated in accordance with the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines. The developed method was implemented to estimate the amount of cyclophosphamide and its related substances. Results: Chromatographical conditions were optimised, and the best chromatographical conditions with adequate resolution for cyclophosphamide and its related substances were achieved using the enable X-Bridge C18 column, using a mobile phase combination of phosphate buffer pH 7.0, water, methanol, and acetonitrile at a flow rate of 0.5 ml/min. The detection was monitored at a wavelength of 200 nm. The developed method was validated for system suitability, specificity, limit of detection (LOD), limit of quantitation (LOQ), linearity, precision, accuracy and robustness. The results indicate that the method was sensitive and could detect and quantify lower levels of cyclophosphamide and its related substances. The linearity range was from LOQ to 150 %, and a correlation coefficient ranging from 0.9987 to 0.9999 indicates that at this concentration range, substances were highly linear. This was further supported by satisfactory forced degradation studies. Conclusion: The developed analytical method is simple, precise, and reproducible and thus can be used for the stability-indicating analysis of cyclophosphamide and its related substances in pharmaceutical formulations.

Determination of vancomycin and meropenem in serum and synovial fluid of patients with prosthetic joint infections using UPLC-MS/MS.

Numerous studies have suggested that intra-articular administration of antibiotics following primary revision surgery may be one of the methods for treating prosthetic joint infection (PJI). Vancomycin and meropenem are the two most commonly used antibiotics for local application. Determining the concentrations of vancomycin and meropenem in the serum and synovial fluid of patients with PJI plays a significant role in further optimizing local medication schemes and effectively eradicating biofilm infections. This study aimed to establish a rapid, sensitive, and accurate ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for determining the concentrations of vancomycin and meropenem in human serum and synovial fluid. Serum samples were processed using acetonitrile precipitation of proteins and dichloromethane extraction, while synovial fluid samples were diluted before analysis. Chromatographic separation was achieved in 6min on a Waters Acquity UPLC BEH C18 column, with the mobile phase consisting of 0.1% formic acid in water (solvent A) and acetonitrile (solvent B). Quantification was carried out using a Waters XEVO TQD triple quadrupole mass spectrometer with an electrospray ionization (ESI) source in positive ion mode. The multiple reaction monitoring (MRM) mode was employed to detect the following quantifier ion transitions: 717.95-99.97 (norvancomycin), 725.90-100.04 (vancomycin), 384.16-67.99 (meropenem). The method validation conformed to the guidelines of the FDA and the Chinese Pharmacopoeia. The method demonstrated good linearity within the range of 0.5-50 μg/ml for serum and 0.5-100 μg/ml for synovial fluid. Selectivity, intra-day and inter-day precision and accuracy, extraction recovery, matrix effect, and stability validation results all met the required standards. This method has been successfully applied in the pharmacokinetic/pharmacodynamic (PK/PD) studies of patients with PJI.

A Validated Chiral Chromatography Method for Enantiomeric Separation of Pomalidomide in Human Plasma.

In the present work, new chiral stationary phase high-performance liquid chromatography (CSP-HPLC) method was established and validated for the quantification of pomalidomide (PMD) enantiomers in human plasma. The chromatographic enantiomeric separation was achieved on a Daicel-CSP, Chiralpack IA 4.6 × 250mm, 5μm; because of its advantages of high degree of retention, high resolution capacity, better reproducibility, ability to produce lower back pressure and low degree of tailing. The mobile phase was maintained as methanol: glacial acetic acid (499.50ml:50μL). Ultraviolet wavelength for detection was 220nm. PMD enantiomer-I and enantiomer-II were separated at 8.83 and 15.34min, respectively. Limit of detection and limit of quantification for each enantiomer and the calibration curve of standard PMD was linear in range between 10-5,000ngmL-1. The method was validated according to The International Council for Harmonisation of Technical Requirements of Pharmaceuticals for Human Use (ICH(Q2R1)) specific guidelines. We found no interference peak with PMD chromatogram obtained. This is a simple, reliable and specific method for detection and quantification of enantiomer of PMD in human plasma sample.