Highly sensitive validated RP-HPLC–DAD method for simultaneous quantification of doxorubicin and docetaxel: augmenting industrial application

Vitamin D (VD) and its metabolites appear in tissues and biological fluids at low concentrations. When combined with complexity of tissue matrices, quantification of VD metabolites in biological samples is challenging. An LC‐MS/MS method for the comparative high throughput quantification of four biologically important VD metabolites was developed using isocratic elution condition coupled with an Atmospheric Pressure Chemical Ionization (APCI) and Multiple Reaction Monitoring (MRM) on triple quadrupole mass spectrometer. To optimize LC ‐ MS/MS conditions, 25‐Hydroxyvitamin D3 (25OH‐D3) was spiked into serum and recovered by liquid‐liquid extraction. Standard curves for all tested analytes displayed liner relationships in the range of 10 ‐ 2500 ng/mL with correlation coefficients of 0.9912 (25OH‐D2), 0.9901 (25OH‐D3), 0.9982 (VD2) and 0.9992 (VD3). Detection limits of quantification (LOQ) for all molecules were determined at 10 ng/mL with signal to noise (s/n) ratios: 10 (25OH‐D2), 14 (25OH‐D3), 8 (VD2), and 14 (VD3) from serum. Relative standard errors of pooled spiked serum samples were less than 3.5%. Future work will focus on extraction from various tissue types.

Developing countries face enormous challenges with substandard and falsified antimalarial drugs. One specific issue is the lack of a simple, cost-effective, and robust HPLC method to simultaneously determine and quantify the active pharmaceutical ingredients (APIs) in fixed-dose artemether-lumefantrine pharmaceutical dosage forms. The current study developed a novel, simple, sensitive, precise, accurate, and cost-effective RP-HPLC method for the simultaneous determination and quantification of artemether and lumefantrine in pharmaceutical dosage forms. The HPLC analysis was carried out on an Agilent 1260 Infinity Series HPLC system equipped with an ODS Intersil-C8 (150 × 4.6 mm) 5.0 µm column, by isocratic elution. The mobile phase composition consisted of acetonitrile and 0.05% orthophosphoric acid buffer of pH 3.5 in the ratio of 70 : 30 v/v. The analysis was performed at a 1 mL/min flow rate and a column temperature of 25°C. The total run time was 6 minutes. The detection was done with a variable wavelength detector (VWD) at an isosbestic point wavelength (λ) of 210 nm. The developed method was validated according to the ICH guidelines concerning system suitability, specificity, linearity, accuracy, precision, and robustness. The system suitability of the developed method revealed satisfactory theoretical plates and symmetry factors. The method proved to be specific, with no interference of mobile phase or excipients. The calibration plot exhibited linearity over the concentration range of 275-1925 μg/mL with R2 = 0.9992 for artemether and a range of 150-1050 μg/mL with R2 = 0.9985 for lumefantrine. The accuracy of the method, determined by the recovery study, was 99.79-100.16% for artemether and 99.04-99.50% for lumefantrine. The % RSD values for intraday precision were 0.175 and 0.203, while interday precision values were 0.340 and 0.554 for artemether and lumefantrine, respectively. The method demonstrated robustness when subjected to slight modifications in the flow rate, column temperature, and mobile phase composition. The developed analytical method proved satisfactory as per ICH guidelines and hence can be used for the determination and quantification of artemether and lumefantrine in bulk drug and pharmaceutical dosage forms.

A method for the simultaneous separation, identification, quantification and confirmation of the presence of 21 glucocorticoids (GCC) in equine plasma by liquid chromatography coupled with triple stage quadrupole tandem mass spectrometry (LC/TSQ-MS/MS) is described. Plasma sample augmented with the 21 GCC was extracted with methyl tert-butyl ether (MTBE) and analyzed by positive electrospray ionization. Desoxymetasone or dichlorisone acetate was used as the internal standard (IS). Quantification was performed by IS calibration. For each drug, one major product ion was chosen and used for screening for that drug. Analyte confirmation was performed by using the three most intense product ions formed from the precursor ion and the corresponding mass ratios. The recovery of the 21 GCC when spiked into blank plasma at 5 ng/mL was 45-200% with coefficient of variation (CV) from 0.3-18%. The limit of detection (LOD) and that of quantification (LOQ) for most of the analytes were 50-100 pg/mL and 1 ng/mL, respectively, whereas that of confirmation (LOC) was 100-300 pg/mL depending on the analyte. Intra- and inter-day precisions expressed as CV for quantification of 1 and 10 ng/mL was 1.0-17%, and 0.51-19%, respectively, and the accuracy was from 84-110%. The linear concentration range for quantification was 0.1-100 ng/mL (r(2) > 0.997). Estimated measurement uncertainty was from 11-37%. This study was undertaken to develop a method for simultaneous screening, identification, quantification and confirmation of these agents in post-race equine plasma samples. The method has been successfully applied to screening of a large number of plasma samples obtained from racehorses in competition and in pharmacokinetic studies of dexamethasone in the horse and concurrent changes in endogenous GCC, hydrocortisone and cortisone. The method is simple, sensitive, selective and reliably reproducible.

Detection and quantification of drugs from various biological matrices are of immense importance in forensic toxicological analysis. Despite the various reported methods, development of a new method for the detection and quantification of drugs is still an active area of research. However, every method and biological matrix has its own limitation, which further encourage forensic toxicologists to develop new methods and to explore new matrices for the analysis of drugs. In this study, an electrospray ionization-liquid chromatograph-tandem mass spectrometry (ESI-LC-MS/MS) method is developed and validated for simultaneous identification and quantification of 24 drugs of forensic relevance in various body fluids, namely, whole blood, plasma and vitreous humour. The newly developed method has been validated for intra-day and inter-day accuracy, precision, selectivity and sensitivity. Absolute recovery shows a mean of 84.5, 86.2, and 103% in the vitreous humour, whole blood and plasma respectively, which is suitable for the screening procedure. Further, the absolute matrix effect (AME) shows a mean of 105, 96.5, and 109% in the vitreous humour, whole blood and plasma, respectively. In addition, to examine the practical utility of this method, it has been applied for screening of drugs in post-mortem samples of the vitreous humour, whole blood and plasma collected at autopsy from ten cadavers. Experimental results show that the newly developed method is well applicable for screening of analytes in all the three matrices.

In this study, we established a comprehensive method for simultaneous identification and quantification of tetrodotoxin (TTX) in fresh pufferfish tissues and pufferfish-based products using liquid chromatography/quadrupole-linear ion trap mass spectrometry (LC-QqLIT-MS). TTX was extracted by 1% acetic acid-methanol, and most of the lipids were then removed by freezing lipid precipitation, followed by purification and concentration using immunoaffinity columns (IACs). Matrix effects were substantially reduced due to the high specificity of the IACs, and thus, background interference was avoided. Quantitation analysis was therefore performed using an external calibration curve with standards prepared in mobile phase. The method was evaluated by fortifying samples at 1, 10, and 100 ng/g, respectively, and the recoveries ranged from 75.8%–107%, with a relative standard deviation of less than 15%. The TTX calibration curves were linear over the range of 1–1 000 μg/L, with a detection limit of 0.3 ng/g and a quantification limit of 1 ng/g. Using this method, samples can be further analyzed using an information-dependent acquisition (IDA) experiment, in the positive mode, from a single liquid chromatography-tandem mass spectrometry injection, which can provide an extra level of confirmation by matching the full product ion spectra acquired for a standard sample with those from an enhanced product ion (EPI) library. The scheduled multiple reaction monitoring method enabled TTX to be screened for, and TTX was positively identified using the IDA and EPI spectra. This method was successfully applied to analyze a total of 206 samples of fresh pufferfish tissues and pufferfish-based products. The results from this study show that the proposed method can be used to quantify and identify TTX in a single run with excellent sensitivity and reproducibility, and is suitable for the analysis of complex matrix pufferfish samples.

Simultaneous voltammetric determination of 7-methyl-guanine and 5-methyl-cytosine using a cathodically pre-treated boron-doped diamond electrode

This paper describes a method for simultaneous extraction and concurrent quantification of metabolites of norepinepohrine (NE) and 5-hydroxytryptamine (5-HT) in human urine using an isocratic HPLC-ECD technique. These metabolites include 4-hydroxy-3-methoxy mandelic acid (VMA), 3-methoxy-4-hydroxyphenylglycol (MHPG), normetanephrine (NMTN), metanephrine (MTN), and 5-hydroxyindoleacetic acid (5-HIAA). The samples of urine were passed through preconditioned alumina-B column and washed with diethylether to remove interfering substances. Metabolites were extracted in ethylacetate. The extracts were evaporated under nitrogen, reconstituted in water and injected into a 5μ spherical, C18, reverse phase column for HPLC-ECD analysis. The analytes were eluted with mobile phase containing citric acid, sodium acetate, sodium octyl sulfate, Na2EDTA, methanol 10%, and isopropanol 0.5%. Isoproterenol was used as an internal standard. Electrochemical detection was carried out at a potential of +0.60 V and a flow rate of 0.5 mL/minute. Peaks were characterized by their retention times. Concentrations were determined by the method using ratio of the peak areas of metabolites to that of internal standard. A linear relationship between the ratio of the areas and concentrations was obtained between 5.0–90 ng/mL of each VMA, MHPG, NMTN, and MTN. The sensitivity for 5-HIAA was found to be ten times higher with a linear relationship between 1.0–8.0 ng/mL. Total elution time for all the metabolites was less than 20 minutes.

Introduction: There is a continuous expansion in number of botanical medicinal products and increase in consumers who often use it. One of such natural source products with versatile traditional uses as treatment for variety of diseases is Moringa oleifera Lam. Objectives: After the pharmacological activity of a new drug candidate is approved, the subsequent development of this product requires substantial information about its physical and chemical properties before designing its dosage form. Methods: Organoleptic properties, physicochemical characteristics, solubility profile, swelling index, partition coefficient, analyses of thermal behaviour, stress stability, heavy metals and microbial limit tests were performed in preformulation studies of 95 % ethanol extract of M. oleifera leaves. Results: The results of preformulation studies illustrated several characteristic properties that should be consider during formulation of Moringa extract. In addition to that, a HPLC-UV method for simultaneous detection and quantification of three reference markers was developed and validated. Conclusion: The 95 % ethanol extract of Moringa leaf is relevant to the development of phytomedicines, dietary supplements or cosmetics. To the best of our knowledge, the present study is the first preformulation study which aimed to determine the physicochemical properties of 95 % ethanol extract of Moringa oleifera leaf.

Citrus, a raw material widely used in food and medicine, is susceptible to fungal infection and its metabolites during growth, transportation, and storage. Thus, monitoring the residual levels of various mycotoxins in Citrus traditional Chinese medicines and related products is crucial. This study described a simple, reliable, and sensitive method for simultaneous identification and quantification of 30 mycotoxins in Citrus products. The method is based on modified quick, easy, cheap, effective, rugged, and safe extraction and purification followed by ultra-high-performance liquid chromatography-quadrupole/orbitrap high-resolution mass spectrometry. The limit of detection ranged from 0.10 to 1.50μg/kg, and the quantification ranged from 0.25 to 5.00μg/kg. The recoveries at three spiked levels were 64.90-99.72% and the relative standard deviation was less than 12%. The method was applied to 55 Citrus samples. The detection rates of tentoxin and mycophenolic acid were the highest, reaching 22.7% and with concentration ranges of 0.33-1.03 and 0.57-2.09μg/kg, respectively. All contamination levels were below the maximum residue limits recommended by the European Commission and China. These results could be used to establish guidelines for screening mycotoxins in Citrus products and the limits of acceptable levels.

The blood-brain barrier (BBB) consists of brain capillary endothelial cells linked by tight junctions and serves to regulate the transfer of endogenous compounds and xenobiotics between the circulating blood and brain interstitial fluid. We have developed a methodology to characterize brain-to-blood efflux transport in vivo, using the Brain Efflux Index and an in vitro culture model of the BBB, i.e., a conditionally immortalized cell line of the neurovascular unit. Employing these methods, we showed that the BBB plays an important role in protecting the brain by transporting neurotransmitters, neuromodulators, metabolites, uremic toxins, and xenobiotics together with atrial natriuretic peptide from the brain interstitial fluid to the circulating blood. We also developed a highly selective, sensitive LC-MS/MS method for simultaneous protein quantification. We found significant species differences in the expression amounts of various BBB transporter proteins among mice, rats, marmosets, cynomolgus monkeys, and humans. Among transporter proteins at the BBB, multidrug resistance protein 1 (Mdr1/Abcb1) is known to generate a concentration gradient of unbound substrate drugs between the blood and brain. Based on measurements of the intrinsic efflux transport rate of Mdr1 and the protein expression amounts of Mdr1 in mouse brain capillaries and Mdr1-expressing cell lines, we predicted the unbound drug concentration gradients of 7 drugs in the mouse brain in vivo. This was the first successful prediction of in vivo drug transport activity from in vitro experimental data and transporter protein concentration in tissues. This methodology and findings should greatly advance central nervous system barrier research.

Development and validation of an enantioselective SFC-MS/MS method for simultaneous separation and quantification of oxcarbazepine and its chiral metabolites in beagle dog plasma.

A method for simultaneous detection and quantification is presented to determine the presence of isoflavones and bisphenol A in a biological sample. A coulometric array detector was used with reversed-phase high-performance liquid chromatography (HPLC). Daidzein (1), glycitein (2), genistein (3) and their glucoside conjugates, daidzin (4), glycitin (5) and genistin (6), were measured as phytochemicals. Also assayed here was equol (7), a metabolite from compound 1, and bisphenol A (8), an industrial chemical that acts as an endocrine disrupter. All chemicals were simultaneously detected by using a 600-mV single detection voltage with high efficacy. A mixture of 1, 3 and 8 was orally administered to rats, and the levels of these three chemicals in the serum were clearly increased after a 4 kU beta-glucuronidase treatment. The levels of compounds 1 and 3 in the serum were detected at 1665 and 2040 ng/ml, while 8 was at a low level of 417 ng/ml. Compound 7 in the serum was not detected until after enzymatic hydrolysis (72 ng/ml). These results suggest that this analytical method would be useful for metabolic and pharmacokinetic studies on isoflavones and bisphenol A.

Objective:The main objective of the study was to "develop and validate a robust Reverse-Phase High-Performance Liquid Chromatography (RP-HPLC) method for the simultaneous estimation of Diosgenin and Piperine in bulk as well as in human plasma samples, with Nintedanibethanesulfonate as an internal standard (IS)". Method:Chromatographic separation was performed on Phenomenex C18 column (250 mm X 4.6 mm, 5µ). Methanol and 0.05% Ortho-phosphoric acid in water in the ratio of 50:50 % V/V was used as the mobile phase at a flow rate of 1 mL/min. Diosgenin and Piperine have different absorption maxima, so the detection was carried out at 210 nm and 343 nm, respectively. In plasma analysis, the method for the extraction was optimized by a protein precipitation extraction method using methanol because it is cost-effective with high drug recovery. Results: Consequently, this method has demonstrated excellent linearity with concentrations ranging from 0.40- 16.00 µg/mL and 0.15-6.00 µg/mL for Diosgenin and Piperine, respectively. Therefore, the retention times were approximately 3.65, 5.62, and 6.52 minutes for the IS, Diosgenin, and Piperine, respectively. On the other hand, the percentage recovery from the extracts was significantly high, as confirmed by the results, which were >92% for both compounds under investigation, specifically Diosgenin, 92.87% and Piperine, 93.09%. Moreover, the precision, as well as accuracy, of all the levels such as LLOQ, LQC, MQC, HQC, were all within Conclusion: A simple, sensitive, and accurate RP-HPLC method for the analysis of Diosgenin and Piperine has been successfully developed. The efficiency of the single internal standard using dual-wavelength detection is impressive, thus making the method suitable for its routine bio-analytical application/pharmacokinetic study for Diosgenin and Piperine.

This thesis reports an integrated analytical and physicochemical approach for the study of natural substances and new drugs based on mass spectrometry techniques combined with liquid chromatography. In particular, Chapter 1 concerns the study of Berberine a natural substance with pharmacological activity for the treatment of hepatobiliary and intestinal diseases. The first part focused on the relationships between physicochemical properties, pharmacokinetics and metabolism of Berberine and its metabolites. For this purpose a sensitive HPLC-ES-MS/MS method have been developed, validated and used to determine these compounds during their physicochemical properties studies and plasma levels of berberine and its metabolites including berberrubine(M1), demethylenberberine(M3), and jatrorrhizine(M4) in humans. Data show that M1, could have an efficient intestinal absorption by passive diffusion due to a keto-enol tautomerism confirmed by NMR studies and its higher plasma concentration. In the second part of Chapter 1, a comparison between M1 and BBR in vivo biodistribution in rat has been studied. In Chapter 2 a new HPLC-ES-MS/MS method for the simultaneous determination and quantification of glucosinolates, as glucoraphanin, glucoerucin and sinigrin, and isothiocyanates, as sulforaphane and erucin, has developed and validated. This method has been used for the analysis of functional foods enriched with vegetable extracts. Chapter 3 focused on a physicochemical study of the interaction between the bile acid sequestrants used in the treatment of hypercholesterolemia including colesevelam and cholestyramine with obeticolic acid (OCA), potent agonist of nuclear receptor farnesoid X (FXR). In particular, a new experimental model for the determination of equilibrium binding isotherm was developed. Chapter 4 focused on methodological aspects of new hard ionization coupled with liquid chromatography (Direct-EI-UHPLC-MS) not yet commercially available and potentially useful for qualitative analysis and for “transparent” molecules to soft ionization techniques. This method was applied to the analysis of several steroid derivatives.

The fixed-dose combination of Amlodipine Besylate (ADB) with Perindopril Tertbutylamine (PTBA) drug is used to treat patients with mild-to-moderate hypertension. In recent times researchers are interested to find the efficient analytical method development and validation for the simultaneous determination of ADB and PTBA in a fixed-dose, film-coated tablet. Therefore, the current study was performed with a reverse-phase liquid chromatography method developed to simultaneously analyze ADB and PTBA in film-coated tablets as fixed-dose combinations. The linearity of the proposed method was calculated by preparing six different mixtures of both ADB and PTBA in the mobile phase. The concentration of both the analytes was analyzed at 56mg/100 mL to 84mg/100 mL and 32mg/100 mL to 48mg/100 mL, respectively. The ratio of acetonitrile and phosphate buffer was 35:65. The flow rate was adjusted to 1.5 ml per minute to reduce the retention time. The validation study was performed for the parameters specificity, linearity, precision, range, limit of detection, limit of quantification, accuracy/biasness, and robustness. The relative percentage standard deviation for Perindopril Tertbutyl amine was 0.148%, and for Amlodipine is 0.312%. These results show that the advanced analysis method for simultaneous analysis of fixed-dose is precise. The theoretical IR spectra were also calculated by Gaussian 9.2 by employing the B3LYP functional at density functional theory (DFT) level study. All these parameters studied in this work authenticate the effectiveness of the developed validation method and ensure its repeatability/reproducibility accordingly. To the best of our knowledge, this is the first time to develop a new fast, and easy method for simultaneous identification and quantification of ADB and PTBA by high-performance liquid chromatography (HPLC) with a time-efficient and cost-effective approach.