Homogeneous liquid-liquid microextraction coupled with HPLC/DAD for determination of nirmatrelvir and ritonavir as COVID-19 combination therapy in human plasma.

The study reports the development of a high-performance liquid chromatography/diode array detection method to measure the levels of nirmatrelvir and ritonavir in human plasma. These two antiviral medications are used for the treatment of COVID-19 and are marketed as Paxlovid®. The method employed sugaring-out induced homogeneous liquid-liquid microextraction to improve sensitivity. Optimization of the method was performed using the one variable at a time approach by adjusting several factors such as type of sugar, extractant, amount of sugar, volume of extractant, and pH of the aqueous sample to achieve the highest efficiency. The developed method was validated according to the Food and Drug Administration guidelines and demonstrated good linearity, accuracy, and precision. The range of linearity was from 1000 to 20,000 ng/mL for nirmatrelvir and 200 to 20,000 ng/mL for ritonavir with correlation coefficient values of 0.998 and 0.996, respectively. Selectivity studies revealed that no others peaks appeared in the retention times of the studied drugs. The stability of nirmatrelvir and ritonavir were also investigated through short term and three cycles of freeze-thaw, and both drugs were found stable. This analytical method could be useful for monitoring drug concentrations in patients undergoing treatment with these medications for COVID-19. In this work, for the first time, SULLME was used for the sensitive determination of nirmatrelvir and ritonavir in biological fluids. The developed method was able to determine both drugs in therapeutic levels with no need to sophisticated techniques like LC-MS. In addition to that, SULLME is considered a simple and green sample preparation in comparison with conventional sample preparation methods.

Analytical Methods for the Determination of Quercetin and Quercetin Glycosides in Pharmaceuticals and Biological Samples.

Flavonoids are plant-derived compounds that have several health benefits, including antioxidative, anti-inflammatory, anti-mutagenic, and anti-carcinogenic effects. Quercetin is a flavonoid that is widely present in various fruits, vegetables, and drinks. Accurate determination of quercetin in different samples is of great importance for its potential health benefits. This review, is an overview of sample preparation and determination methods for quercetin in diverse matrices. Previous research on sample preparation and determination methods for quercetin are summarized, highlighting the advantages and disadvantages of each method and providing insights into recent developments in quercetin sample treatment. Various analytical techniques are discussed including spectroscopic, chromatographic, electrophoretic, and electrochemical methods for the determination of quercetin and its derivatives in different samples. UV-Vis (Ultraviolet-visible) spectrophotometry is simple and inexpensive but lacks selectivity. Chromatographic techniques (HPLC, GC) offer selectivity and sensitivity, while electrophoretic and electrochemical methods provide high resolution and low detection limits, respectively. The aim of this review is to comprehensively explore the determination methods for quercetin and quercetin glycosides in diverse matrices, with emphasis on pharmaceutical and biological samples. The review also provides a theoretical basis for method development and application for the analysis of quercetin and quercetin glycosides in real samples.

A hybrid microcrystalline cellulose/metal-organic framework for dispersive solid phase microextraction of selected pharmaceuticals: A proof-of-concept.

Solid phase microextraction (SPME) is considered simple, ecofriendly, sustainable, cost-effective and timesaving sample preparation mode in comparison with other sample preparation procedures. The researchers always try to develop new sorbents with higher surface area in comparison with other conventional sorbents aiming to enhance the extraction efficiency. In this work for the first time, a comparative study was performed between Ca-BTC MOF (1,3,5-benzenetricarboxylic acid, BTC; metal-organic framework, MOF) and a hybrid Ca-BTC-MCC MOF (microcrystalline cellulose, MCC) by using as model compounds seven drugs with different physicochemical properties. The evaluation of the extraction efficiency of both sorbents were obtained by means of an HPLC/DAD instrument configuration in reversed phase mode under isocratic elution mode. The results indicate that Ca-BTC MOF showed superior extraction efficiency than Ca-BTC-MCC MOF in the case of all analytes except nirmatrelvir and ritonavir. The results highlight that not only the surface area of adsorbents controlled the adsorption capacity, but also other factors have a role in extraction efficiency including morphology of adsorbent and physico-chemical properties of the analytes. It is worth mentioning that this is the first time that a comparative study was performed between Ca-BTC MOF and Ca-BTC-MCC MOF hybrid material.

The anti-COVID-19 drug Favipiravir: Degradation, Method development, Validation, NMR/LC-MS characterization, and In-vitro safety evaluation.

It is critical to characterize the degradation products of therapeutic drugs to determine their safety as these degradation products may possess fatal effects on the human physiological system. Favipiravir (FVP), a novel anti-Covid-19 drug, that is recently used all over the world with a great impact on humanity was our target to explore more about its toxicity, the margins of its safety, and its degradants in different degradation conditions. The goal of this study is to identify, characterize, and confirm the structures of FVP oxidative and alkaline breakdown products, as well as to assess their safety utilizing in-vitro SRB cytotoxicity assay on normal human skin fibroblasts (NHSF) cell lines. After oxidative and alkaline degradation of FVP, one degradation product was produced in each condition which was isolated from FVP using flash chromatography, characterized by 1HNMR and LC-MS/MS techniques. A reversed-phase Thermo Fischer Hypersil C18 column (4.6 × 150 mm, 5 m) was used to achieve HPLC chromatographic separation. Acetonitrile-5 mM potassium dihydrogen phosphate (pH 2.5) (50:50, v/v) was employed as the mobile phase, with a flow rate of 1 mL/min. At 332 nm, the column effluent was measured. Over the concentration range of 0.5-100 µg/mL, the calibration curve was linear. The intra-day and inter-day relative standard deviations were less than 2%, and good percentage recoveries were obtained that fulfilled the acceptance criteria of the International Conference on Harmonization (ICH) recommendations. The Plackett-Burman design was used to assess the robustness. Each degradant was isolated single using Flash chromatography and methylene chloride: methanol gradient mobile phase. The chemical structures of the degradation products have been confirmed and compared to the intact FVP using 1H-NMR, and Mass spectroscopy. A postulated mechanism of the degradation process has been depicted and the degradants fragmentation pattern has been portrayed. In addition, the in vitro SRB cytotoxicity assay to evaluate the safety profile of FVP and the degradation end products showed their high safety margin in both conditions with IC50 ˃100 µg/ml with no signs of toxicity upon examination of the treated NHSF cells under the optical microscope.

A gadolinium-based magnetic ionic liquid for supramolecular dispersive liquid-liquid microextraction followed by HPLC/UV for the determination of favipiravir in human plasma.

Favipiravir is a potential antiviral medication that has been recently licensed for Covid-19 treatment. In this work, a gadolinium-based magnetic ionic liquid was prepared and used as an extractant in dispersive liquid-liquid microextraction (DLLME) of favipiravir in human plasma. The high enriching ability of DLLME allowed the determination of favipiravir in real samples using HPLC/UV with sufficient sensitivity. The effects of several variables on extraction efficiency were investigated, including type of extractant, amount of extractant, type of disperser and disperser volume. The maximum enrichment was attained using 50 mg of the Gd-magnetic ionic liquid (MIL) and 150 µl of tetrahydrofuran. The Gd-based MIL could form a supramolecular assembly in the presence of tetrahydrofuran, which enhanced the extraction efficiency of favipiravir. The developed method was validated according to US Food and Drug Administration bioanalytical method validation guidelines. The coefficient of determination was 0.9999, for a linear concentration range of 25 to 1.0 × 105  ng/ml. The percentage recovery (accuracy) varied from 99.83 to 104.2%, with RSD values (precision) ranging from 4.07 to 11.84%. The total extraction time was about 12 min and the HPLC analysis time was 5 min. The method was simple, selective and sensitive for the determination of favipiravir in real human plasma.

Development and validation of a simple and sensitive LC-MS/MS method for the quantification of cefazolin in human plasma and its application to a clinical pharmacokinetic study.

Cefazolin is widely used during surgery to prevent surgical site infections (SSIs). Although cefazolin redosing is often needed due to its short half-life, the appropriate redosing schedule remains controversial and there is limited information on cefazolin disposition following repeated doses during surgery. In parallel with an ongoing cefazolin redosing clinical study, we have developed and fully validated a simple and robust liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of cefazolin in human plasma. A simple protein precipitation was used for sample preparation. MS/MS analysis was performed using multiple reaction monitoring (MRM) under a positive ionization mode. The lower limit of quantification (LLOQ) for cefazolin was evaluated at 0.25 µg/mL and a linearity ranging from 0.25 to 300 µg/mL. Accuracy was ≤ 114.3% for quality controls and ≤ 118.2% for LLOQ; intra-day and inter-day precision ranging from 1.9% to 14.2% for all quality controls and LLOQ. Matrix effect, extraction recovery, stability testing, dilution integrity, hemolysis effects and whole blood stability have all been investigated. A total of 17 parameters were validated and passed their validation criteria. The method was applied in the quantification of cefazolin in clinical plasma samples and was able to successfully determine the concentrations in patients undergoing various surgeries. In comparison with other prior published methods, our method has a simple sample preparation combined with a short analysis run time, a wide dynamic range and low limit of quantification, and is a fully validated assay that abides by FDA guidance.

Determination of favipiravir in human plasma using homogeneous liquid-liquid microextraction followed by HPLC/UV.

Background: Favipiravir is an antiviral drug that was recently approved for the management of COVID-19 infection. Aim: This work aimed to develop a new method, using sugaring-out induced homogeneous liquid-liquid microextraction followed by HPLC/UV for the determination of favipiravir in human plasma. Materials & methods: The optimum extraction conditions were attained using 500 µl of tetrahydrofuran as an extractant and 1400 mg of fructose as a phase-separating agent. Results: The developed method was validated according to the US FDA bioanalytical guidelines and was found linear in the range of 25-80,000 ng/ml with a correlation coefficient of 0.999. Conclusion: These results showed that the developed method was simple, easy, valid and adequately sensitive for determination of favipiravir in plasma for bioequivalence studies.

Menthol-assisted homogenous liquid-liquid microextraction for HPLC/UV determination of favipiravir as an antiviral for COVID-19 in human plasma.

Favipiravir is a promising antiviral agent that has been recently approved for treatment of COVID-19 infection. In this study, a menthol-assisted homogenous liquid-liquid microextraction method has been developed for favipiravir determination in human plasma using HPLC/UV. The different factors that could affect the extraction efficiency were studied, including extractant type, extractant volume, menthol amount and vortex time. The optimum extraction efficiency was achieved using 300 µL of tetrahydrofuran, 30 mg of menthol and vortexing for 1 min before centrifuging the sample for 5 min at 3467g. Addition of menthol does not only induce phase separation, but also helps to form reverse micelles to facilitate extraction. The highly polar favipiravir molecules would be incorporated into the hydrophilic core of the formed reverse micelle to be extracted by the non-polar organic extractant. The method was validated according to the FDA bioanalytical method guidelines. The developed method was found linear in the concentration range of 0.1 to 100 µg/mL with a coefficient of determination of 0.9992. The method accuracy and precision were studied by calculating the recovery (%) and the relative standard deviation (%), respectively. The recovery (%) was in the range of 97.1-103.9%, while the RSD (%) values ranged between 2.03 and 8.15 %. The developed method was successfully applied in a bioequivalence study of Flupirava® 200 mg versus Avigan® 200 mg, after a single oral dose of favipiravir administered to healthy adult volunteers. The proposed method was simple, cheap, more eco-friendly and sufficiently sensitive for biomedical application.

Homogeneous liquid-liquid extraction as an alternative sample preparation technique for biomedical analysis.

Liquid-liquid extraction is a widely used technique of sample preparation in biomedical analysis. In spite of the high pre-concentration capacities of liquid-liquid extraction, it suffers from a number of limitations including time and effort consumption, large organic solvent utilization, and poor performance in highly polar analytes. Homogeneous liquid-liquid extraction is an alternative sample preparation technique that overcomes some drawbacks of conventional liquid-liquid extraction, and allows employing greener organic solvents in sample treatment. In homogeneous liquid-liquid extraction, a homogeneous phase is formed between the aqueous sample and the water-miscible extractant, followed by chemically or physically induced phase separation. To form the homogeneous phase, aqueous samples are mixed with water-miscible organic solvents, water-immiscible solvents/cosolvents, surfactants, or smart polymers. Then, phase separation is induced chemically (adding salt, sugar, or buffer) or physically (changing temperature or pH). This mode is rapid, sustainable, and cost-effective in comparison with other sample preparation techniques. Moreover, homogeneous liquid-liquid extraction is more suitable for the extraction of delicate macromolecules such as enzymes, hormones, and proteins and it is more compatible with liquid chromatography with tandem mass spectrometry, which is a vital technique in metabolomics and proteomics. In this review, the principle, types, applications, automation, and technical aspects of homogeneous liquid-liquid extraction are discussed.

Structure- and Ligand-Based in silico Studies towards the Repurposing of Marine Bioactive Compounds to Target SARS-CoV-2.

This work was a structured virtual screening for marine bioactive compounds with reported antiviral activities which were subjected to structure-based studies against SARS-CoV-2 co-crystallized proteins. The molecular docking of marine bioactive compounds against the main protease (Mpro, PDB ID: 6lu7 and 6y2f), the spike glycoprotein (PDB ID: 6vsb), and the RNA polymerase (PDB ID: 6m71) of SARS-CoV-2 was performed. Ligand-based approach with the inclusion of rapid overlay chemical structures (ROCS) was also addressed in order to examine the probability of these marine compounds sharing relevance and druggability with the reported drugs. Among the examined marine library, the highest scores in different virtual screening aspects were displayed by compounds with flavonoids core, acyl indole, and pyrrole carboxamide alkaloids. Moreover, a complete overlay with the co-crystallized ligands of Mpro was revealed by sceptrin and debromo-sceptrin. Thalassoilin (A-B) which was found in the Red Sea exhibited the highest binding and similarity outcomes among all target proteins. These data highlight the importance of marine natural metabolites in regard to further studies for discovering new drugs to combat the COVID-19 pandemic.

Enantioselective Separation of Chiral N1-Substituted-1H-pyrazoles: Greenness Profile Assessment and Chiral Recognition Analysis.

Two commercialized polysaccharide-based chiral stationary phases, Lux cellulose-2 and Lux amylose-2, were examined for their chiral recognition ability on a set of 18 biologically active racemic 4,5-dihydro-1H-pyrazole derivatives by applying normal and polar organic elution modes. The results showed that all compounds were baseline-resolved with at least one of the used elution modes. The cellulose-based column was superior using polar organic mobile-phase compositions with analysis times close to 5 min and resolutions up to 18, while the enantiomer-resolving ability of amylose-based columns was greater using the normal elution mode with analysis times close to 30 min and resolutions up to 30. The competition between the analytes and the mobile phase constituents on H-bond interactions with the stationary phase has been discussed, and the impact of this competition on chiral recognition has been investigated. It was found that the polar organic mode is very beneficial for short run times and sharp peaks. The developed enantioselective high-performance liquid chromatography (HPLC) methods will be applied to monitor the stereoselective synthesis of compounds 1-18 or to develop preparative HPLC techniques for compounds 1-18, followed by stereospecific pharmacological studies for each enantiomer separately. Greenness profile assessment of the different elution solvents was carried out using the AGREE metric approach.

Sugaring-out induced homogeneous liquid-liquid microextraction as an alternative mode for biological sample preparation: A comparative study.

Miniaturization of liquid-liquid extraction is a growing field of sample preparation to reduce solvent consumption, protect the environment, and preserve operators' health. In this work, four different modes of liquid-liquid microextraction have been compared including dispersive liquid-liquid microextraction, binary and ternary salting-out, and sugaring-out induced liquid-liquid microextraction. The extraction efficiency was evaluated by the enrichment factors of 14 different drugs from three pharmacological classes. Compared with the other modes, sugaring-out induced liquid-liquid microextraction was found to be the most efficient and, thus, it was applied for sample preparation of the antivirals in human plasma. Method optimization was performed using response surface methodology for the sugar type and amount (in mg), the sample pH, the equilibration time (in min), and the extractant volume (in µL). The method was then validated and found linear in the concentration range of 0.10-10 µg/mL for daclatasvir, 0.05-10 µg/mL for velpatasvir, and 0.20-10 µg/mL for ledipasvir, with correlation coefficients in the range 0.996-0.999. These results shows that sugaring-out induced liquid-liquid microextraction could be a more efficient microextraction mode for preparation of biological samples. Compared with other types of microextraction, sugaring-out induced liquid-liquid microextraction is greener, simpler, and cost-effective, with less tendency to affect the sample pH.

Salting-out induced liquid-liquid microextraction for alogliptin benzoate determination in human plasma by HPLC/UV.

Salting-out induced liquid-liquid microextraction method has been developed for plasma sample treatment before determination of alogliptin by high performance liquid chromatography with UV detection. Several parameters were optimized to achieve maximum enrichment, including type of extractant, volume of extractant, type of anion, type of cation, salt amount and pH. The optimum conditions were attained using 500 µL of acetonitrile, added to 1 mL of aqueous sample containing 250 mg of sodium chloride at pH 12. An RP-HPLC method was developed and validated according to the International Conference on Harmonization guidelines M10. The method was linear in the concentration range of 0.1 to 50 µg/mL (correlation coefficient = 0.997). The limit of detection was 0.019 µg/mL and limit of quantitation was 0.06 µg/mL. The method was accurate and precise with an average % recovery of 99.7% and a % relative standard deviation ranging between 1.5 and 2.5. These results showed that the salting-out induced liquid-liquid microextraction methods could be better than other sample preparation protocols in terms of sensitivity, easiness, solvent consumption and waste reduction.

Lipid polymer hybrid nanocarriers as a combinatory platform for different anti-SARS-CoV-2 drugs supported by computational studies.

The COVID-19 pandemic caused by SARS-CoV-2 has demonstrated the potential of emergent pathogens to severely damage public health and global economies. As a consequence of the pandemic, millions of people have been forced into self-isolation, which has negatively affected the global economy. More efforts are needed to find new innovative approaches that could fundamentally change our understanding and management of this disaster. Herein, lipid polymer hybrid nanoparticles (LPH NPs) were utilized as a platform for the delivery of azithromycin or niclosamide in combination with piroxicam. The obtained systems were successfully loaded with both azithromycin and piroxicam (LPHAzi-Pir) with entrapment efficiencies (EE%) of 74.23 ± 8.14% and 51.52 ± 5.45%, respectively, or niclosamide and piroxicam (LPHNic-Pir) with respective EE% of 85.14 ± 3.47% and 48.75 ± 4.77%. The prepared LPH NPs had a core-shell nanostructure with particle size ≈ 125 nm and zeta potential ≈ -16.5 irrespective of drug payload. A dose-dependent cellular uptake of both LPH NPs was observed in human lung fibroblast cells. An enhanced in vitro antiviral efficacy of both LPHAzi-Pir and LPHNic-Pir was obtained over the mixed solution of the drugs. The LPH NPs of azithromycin or niclosamide with piroxicam displyed a promising capability to hinder the replication of SARS-CoV-2, with IC50 of 3.16 and 1.86 µM, respectively. These results provide a rationale for further in vivo pharmacological as well as toxicological studies to evaluate the potential activity of these drugs to combat the COVID-19 outbreak, especially the concept of combination therapy. Additionally, the molecular docking of macrolide bioactive compounds against papain-like protease (PDB ID:6wuu) was achieved. A ligand-based study, especially rapid overlay chemical structure (ROCS), was also examined to identify the general pharmacophoric features of these compounds and their similarity to reported anti-SARS-CoV-2 drugs. Molecular dynamic simulation was also implemented.

Identification, isolation, structural characterization, in silico toxicity prediction and in vitro cytotoxicity assay of simeprevir acidic and oxidative degradation products.

Simeprevir is a new direct-acting antiviral drug used for the treatment of chronic hepatitis C. In this work, a simple, fast and economical chromatographic method was developed for the determination of simeprevir in the presence of its acidic and oxidative degradation products. The stress studies performed herein showed that simeprevir degraded under acidic and oxidative conditions but was stable under thermal and alkaline conditions. Chromatographic separation was achieved on a reversed-phase Eclipse XDB C18 column (4.6 × 150 mm, 5 µm). The mobile phase consisted of methanol-0.05 M ammonium acetate (pH 4) (90 : 10, v/v) and was used at a flow rate of 1 mL min-1. The column effluent was monitored at 237 nm. The calibration curve was linear over the concentration range of 0.1-20 µg mL-1. The relative standard deviations for the intra-day and inter-day precision were less than 2%, and good percentage recoveries that met the acceptance criteria of the International Conference on Harmonization (ICH) guidelines were obtained. The robustness was assessed using the Plackett-Burman design. The simeprevir degradation products were isolated by flash chromatography and confirmed by 1H NMR and LC-MS/MS techniques. The fully validated chromatographic method can be applied as a stability-indicating method for simeprevir and for routine analysis during quality control. Additionally, in silico toxicity prediction of the degradation products demonstrated a hepatotoxicity alert for DP 1, DP 2, DP 4 and DP 5 and a carcinogenicity alert for DP 3. In view of safety aspects, an in vitro cytotoxicity assay was carried out for simeprevir degradation products. They were found to be non-toxic in vitro at the tested concentrations.

Determination of flibanserin in the presence of confirmed degradation products by a third derivative emission spectrofluorometric method: Application to pharmaceutical formulation.

Flibanserin is a new drug used for the treatment of hypoactive sexual desire disorder. This work is considered the first study concerning the fluorimetric behaviour of flibanserin and its new florescent degradation products. A fast, cost-effective, stability-indicating spectrofluorometric method was developed and validated for the determination of flibanserin in the presence of oxidative degradation products. Stability studies are performed to predict the behaviour of substances under various harsh conditions. Thus, flibanserin was subjected to degradation using hydrogen peroxide. The stability-indicating method was developed and validated per ICH guidelines; it was linear in the range of 0.1-3 µg/mL. The method was accurate and precise as it showed good recoveries between 98.50 and 100.90% and relative standard deviation less than 2%, respectively, and no significant differences were found after statistical comparison with the in-house HPLC method. In addition, the structures of the oxidative degradation products were confirmed using infrared spectroscopy and mass spectrometry, and the proposed degradation pathway was predicted.

LC-MS determination of fentanyl in human serum and application to a fentanyl transdermal delivery pharmacokinetic study.

AIM: Fentanyl is an opioid agonist used for acute and chronic pain management. In this report, a highly sensitive and simple LC-MS/MS method using Hydrophilic Interaction Chromatography (HILIC) column was validated and used for fentanyl quantification in human serum. RESULTS: The isocratic mobile phase was composed of acetonitrile: 10 mM ammonium formate buffer (pH = 3.2; 90:10, v/v). The assay was linear over a concentration range of 10-10,000 pg/ml. The accuracy of the validation method ranged from 93.2 to 107%, and the precision was within 6.4%. Fentanyl was stable during short- and long-term storage. CONCLUSION: The assay has been successfully applied to serum samples obtained from healthy subjects of a fentanyl transdermal pharmacokinetic study.

Precise simultaneous quantification of methadone and cocaine in rat serum and brain tissue samples following their successive i.p. administration.

A sensitive high-performance liquid chromatography (HPLC) assay with dual UV detection has been developed and validated for the simultaneous quantification of methadone and cocaine in rat serum and brain tissue samples. Liquid-liquid extraction using hexanes was applied for samples extraction with Levo-Tetrahydropalmatine (L-THP) as the internal standard. Chromatographic separation of the analytes was achieved on a reversed-phase Waters Symmetry® C18 column (150mm×4.6mm, 5µm). A gradient elution was employed with a mobile phase consisting of 5mM potassium phosphate containing 0.1% triethylamine (pH=6.5) (A) and acetonitrile (B) with a flow rate of 1mL/min. UV detection was employed at 215nm and 235nm for the determination of methadone and cocaine, respectively. The calibration curves were linear over the range of 0.05-10µg/mL for both methadone and cocaine. The assay was validated according to FDA guidelines for bioanalytical method validation and results were satisfactory and met FDA criteria. Inter-day accuracy values of serum and brain samples ranged from 96.97 to 105.59% while intra-day accuracy values ranged from 91.49 to 111.92%. Stability assays showed that both methadone and cocaine were stable during sample storage, preparation, and analytical procedures. The method was successfully used to analyze biological samples obtained from a drug- drug interaction pharmacokinetics (PK) study conducted in rats to investigate the effect of methadone on cocaine PK. Our method not only can be used for bioanalysis of samples obtained from rats but also can potentially be applied to human biological serum samples to monitor compliance to methadone maintenance therapy (MMT) and to detect possible cocaine-methadone co-abuse.

Development and validation of a high performance liquid chromatography quantification method of levo-tetrahydropalmatine and its metabolites in plasma and brain tissues: application to a pharmacokinetic study.

Levo-tetrahydropalmatine (l-THP) is an alkaloid isolated from Chinese medicinal herbs of the Corydalis and Stephania genera. It has been used in China for more than 40 years mainly as an analgesic with sedative/hypnotic effects. Despite its extensive use, its metabolism has not been quantitatively studied, nor there a sensitive reliable bioanalytical method for its quantification simultaneously with its metabolites. As such, the objective of this study was to develop and validate a sensitive and selective HPLC method for simultaneous quantification of l-THP and its desmethyl metabolites l-corydalmine (l-CD) and l-corypalmine (l-CP) in rat plasma and brain tissues. Rat plasma and brain samples were processed by liquid-liquid extraction using ethyl acetate. Chromatographic separation was achieved on a reversed-phase Symmetry® C18 column (4.6 × 150 mm, 5 µm) at 25°C. The mobile phase consisted of acetonitrile-methanol-10 mm ammonium phosphate (pH 3) (10:30:60, v/v) and was used at a flow rate of 0.8 mL/min. The column eluent was monitored at excitation and emission wavelengths of 230 and 315 nm, respectively. The calibration curves were linear over the concentration range of 1-10,000 ng/mL. The intra- and interday reproducibility studies demonstrated accuracy and precision within the acceptance criteria of bioanalytical guidelines. The validated HPLC method was successfully applied to analyze samples from a pharmacokinetic study of l-THP in rats. Taken together, the developed method can be applied for bioanalysis of l-THP and its metabolites in rodents and potentially can be transferred for bioanalysis of human samples.

A fully validated LC-MS/MS method for simultaneous determination of nicotine and its metabolite cotinine in human serum and its application to a pharmacokinetic study after using nicotine transdermal delivery systems with standard heat application in adult smokers.

A sensitive and simple liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for simultaneous determination of nicotine and its main metabolite cotinine in human serum samples. Liquid-liquid extraction using ethyl acetate was employed for serum sample extractions. Chromatographic separation was achieved on Phenomenex Luna(®) HILIC column (150 mm x 3.0mm, 5 µm). Isocratic elution was performed using acetonitrile:100mM ammonium formate buffer (pH=3.2) (90:10, v/v) as the mobile phase, at a flow rate of 0.4 mL/min. Tandem mass spectrometric detection was employed at positive electrospray ionization in MRM mode for the determination of both nicotine and cotinine and their stable isotope labeled internal standards. Analysis was carried out in 8 min over a concentration range of 0.26-52.5 ng/mL and 7.0-1500 ng/mL for nicotine and cotinine, respectively. The assay was validated according to FDA guidelines for bioanalytical method validation and satisfactory results were obtained; the accuracy ranged between 93.39% and 105.73% for nicotine and between 93.04% and 107.26% for cotinine. No significant matrix effect was observed. Stability assays indicated both nicotine and cotinine were stable during sample storage, preparation and analytical procedures. The method was successfully applied to biological samples obtained from a pharmacokinetic study conducted in adult smokers to investigate heat effect on nicotine and cotinine serum levels after nicotine transdermal delivery system (TDS) application.