Simple HPLC-UV Method Research Articles

Ceftobiprole quantification in human serum by HPLC-UV to implement routine TDM in clinical practice

Background and aimsCeftobiprole is a recent 5th generation parenteral cephalosporin with antibacterial activity against a large range Gram+ and Gram− bacteria. Therapeutic drug monitoring (TDM) is an essential tool for maintaining plasma concentrations of antibiotics above the MIC by the end of the dosing interval, thus preventing the resistant strain diffusion. TDM is already recommended for other cephalosporins, and it is a reasonable tool contributing to the safety and efficacy of these drugs. During the treatment of patients in real-life, a number of pharmacokinetic (PK) changes not normally seen in healthy volunteers can occur which can impair the pharmacokinetic/pharmacodynamic target attainment.We aimed to develop simple and rapid HPLC-UV method for determination of ceftobiprole in human serum to implement TDM in clinical practice and support PKs and pharmacokinetic/pharmacodynamic (PK/PD) studies. Materials and methodsSamples preparation of calibration standards, QC, and anonymous patients serum samples was performed by protein precipitation by adding 0.01 ml of sulphosalicylic acid at 30 % to 0.1 ml of each sample. Then samples were vortexed and the centrifuged at 12,000 rpm for 10 min at 4 °C. Fifty microlitres of clear supernatant were diluted 1:1 with mobile phase and transferred into HPLC autosampler held at 8 °C. Chromatographic separation was carried out in a gradient mode at 35 °C on an ultra-Biphenyl column using a Thermo Scientific chromatographic system with a Diode array. Data management was performed with Chromeleon 7.4 software. ResultsThe HPLC-UV method proved to be linear over wide concentration ranges (0.5–50.0 mg/L) and was accurate and reproducible in the absence of matrix effects, allowing for robust, specific, and rapid quantification of ceftobiprole from a low amount of serum (0.1 mL). The mean steady state Ctrough and Cend values measured in the anonymous patients’ samples were 6.26 ± 3.81 mg/L and 22.56 ± 15.69 mg/L, respectively. ConclusionsWe report a broadened simple and fast HPLC with UV detection method for quantification of ceftobiprole in human serum to implement ceftobiprole TDM as clinical routine, and support future (PK/PD) studies in special patients’ population.

Development and Validation of a Simple and Reliable HPLC-UV Method for Determining Gemcitabine Levels: Application in Pharmacokinetic Analysis.

Background and Objectives: Gemcitabine has been used to treat various solid cancers, including, since 1997, metastatic pancreatic cancer. Here, we developed an HPLC-UV method to determine serum gemcitabine levels and use it in pharmacokinetic studies. Materials and Methods: The analysis was performed after a single protein precipitation step on a reversed-phase column, isocratically eluted with sodium phosphate buffer and methanol. For the pharmacokinetic study, NOD/SCID mice received a single dose of gemcitabine at 100 mg/kg by either subcutaneous (SC) or intraperitoneal (IP) administration. Blood samples were collected at 5, 15, and 30 min and 1, 2, 4, and 6 h after the administration of gemcitabine for further analysis. Results: The duration of the analysis was ~12.5 min. The calibration curve was linear (r2 = 0.999) over the range of 1-400 μM. The mean recovery of GEM was 96.53% and the limit of detection was 0.166 μΜ. T1/2, Tmax, Cmax, AUC0-t, and clearance were 64.49 min, 5.00 min, 264.88 μmol/L, 9351.95 μmol/L*min, and 0.0103(mg)/(μmol/L)/min, respectively, for the SC administration. The corresponding values for the IP administration were 59.34 min, 5.00 min, 300.73 μmol/L, 8981.35 μmol/L*min and 0.0108(mg)/(μmol/L)/min (not statistically different from the SC administration). Conclusions: A simple, valid, sensitive, and inexpensive method for the measurement of gemcitabine in serum has been developed. This method may be useful for monitoring gemcitabine levels in cancer patients as part of therapeutic drug monitoring.

A simple and rapid HPLC-UV method for the determination of valproic acid in human plasma using microwave-assisted derivatization with phenylhydrazine hydrochloride

This study presents an efficient high-performance liquid chromatography with ultraviolet detection (HPLC-UV) method for monitoring valproic acid (VPA) level in human plasma. This method is distinguished by its simplicity, cost-effectiveness, and rapid execution, addressing the limitations associated with other advanced analytical techniques like liquid chromatography-mass spectrometry (LC-MS), gas chromatography-mass spectrometry (GC-MS), and immunoassays, which are generally complex and costly for routine application. A challenge in analyzing VPA is its non-linear protein binding profile and the absence of a chromophore in its structure, making direct detection difficult. To overcome this, the study developed an efficient HPLC-UV for VPA determination in human plasma, utilizing a simplified and rapid microwave-assisted derivatization process. Due to the lack of a chromophore in VPA structure, this work developed a microwave-assisted derivatization of VPA using phenylhydrazine hydrochloride (PH HCl). The process optimization was achieved at 450 W for 50 s, facilitating effective HPLC-UV detection. The derivatized product was characterized using 1H nuclear magnetic resonance (NMR) and Fourier transform infrared spectrometer (FT-IR). The derivative, identified as (Z)-N-phenyl-2-propylpentanehydrazonic acid, demonstrated specificity in plasma analysis with no detectable interference. The method exhibited a linear response for VPA concentrations ranging from 30 to 150 μg/mL, with a correlation coefficient exceeding 0.99. Recovery varied between 86.7% and 107%, with a maximum coefficient of variation (CV) of 10.0%. The findings suggest that the microwave-assisted derivatization technique substantially improves the feasibility and cost-effectiveness of HPLC-UV for the analysis of VPA in plasma. This method provides a viable alternative to conventional HPLC methodologies, offering a balance of efficiency and economic practicality for VPA quantification.

Development of Simple HPLC-UV Method for the Simultaneous Determination of Repaglinide, Dexamethasone, and Remdesivir, and its Application to Synthetic Mixture and Human Plasma

Background:: The onset of the COVID-19 pandemic caused numerous difficulties in the treatment of cardiovascular diseases and diabetes mellitus. A persistent risk of developing severe complications and increased mortality from the COVID-19 infection has been reported. In the clinical studies, patients receiving remdesivir and dexamethasone as COVID-19 combination therapy simultaneously with some type II diabetes therapeutic regimens had been reported to have a considerably better state and recover faster. Unfortunately, there is not enough information on the combination of meglitinides, remdesivir, and dexamethasone, and therefore, careful monitoring of the patients' everyday health condition is needed. Objectives:: The present study aimed to describe a high-performance liquid chromatographic method for the determination of repaglinide, dexamethasone, and remdesivir in laboratoryprepared mixtures and human plasma by UV detection. Methods:: Isocratic elution of the mobile phase (consisting of 0.1% trifluoroacetic acid in water and acetonitrile in the ratio 70:30 v/v) was set at a flow rate of 1.0 ml/min, and the developed analytical procedure has been found to be fast and simple. Chromatographic determination was performed on a Purospher® RP – 18 column at room temperature and a UV detector was set at 235 nm. result: The developed method was validated for linearity in the range 2-32 μg/ml. Calibration curves were linear over the selected range with correlation coefficients (R2) greater than 0.996. The coefficients of variation for intraday and interday assay were <2% and the recovery percentages from plasma ranged from 93.83 to 106.49%. Conclusion:: The developed effective and specific method can be applied in routine quality control and clinical laboratory practice.

Validated HPLC-UV method for amphotericin B quantification in a critical patient receiving AmBisome and treated with extracorporeal replacement therapies.

Amphotericin B (AMB) is a polyene macrolide antifungal agent used for treating invasive fungal infections. Liposomal AMB is a lipid dosage form, available as AmBisome, which reduces the toxicity of the drug. A simple HPLC-UV method was developed for the determination of AMB in plasma to study its pharmacokinetic profile in a critical patient receiving AmBisome and treated with extracorporeal replacement therapies. Sample preparation was performed using plasma deproteinization and drug release from liposome by the addition of acetonitrile (ACN)/zinc sulfate and ultrasonication. Chromatographic separation was performed using a C18 column and a mobile phase consisting of phosphate buffer (pH 3.0)/ACN (65/35, v/v). The UV detector was set at 407 nm. The total run time analysis was 23 min. The method was validated according to the standard guidelines and applied to study the pharmacokinetics of AMB in a critical patient. The total run time analysis obtained was shorter than that of the previously reported methods, being useful for therapeutic drug monitoring or pharmacokinetic profile research.

Development and Validation of a Simple, Fast, and Accessible HPLC-UV Method for Cannabinoids Determination in Cannabis sativa L. Extracts and Medicinal Oils.

Cannabis sativa L. is a well-recognized medicinal plant. Cannabis regulations in Argentina are insufficient to solve the problem of patient access to full-spectrum cannabis-based products. So, the market of artisanal products with unknown quality and dosage of cannabinoids is increasing, and so is the local demand and need for analyzing these products. However, much of the latest validated methodologies for cannabinoid quantification include expensive instrumentation that is not always available in laboratories of health institutions in Argentina. The aim of this work was to develop and validate a simple and rapid HPLC-UV method for the identification and quantification of principal cannabinoids in cannabis resins, inflorescences, and medicinal oils using standard HPLC equipment. The cannabinoids selected for validation were cannabidiol acid (CBDA), cannabigerol (CBG), cannabidiol (CBD), cannabinol (CBN), delta-9-tetrahydrocannabinol (Δ9-THC), cannabichromene (CBC), and tetrahydrocannabinol acid (THCA). A method for the simultaneous identification and quantification of these 7 main cannabinoids was developed and then validated. Some data parameters were comparable to other reports with more sophisticated analytical instruments for the analysis of cannabis. The assessed limits of detection and the limits of quantitation ranged from 0.9 to 3.66 μg/mL and 2.78 to 11.09 μg/mL, respectively. The concentration-response relationship of the method indicated a linear relationship between the concentration and peak area with R2 values of > 0.99 for all 7 cannabinoids. The relative standard deviation (RSD%) varied from 2.34 to 4.82 for intraday repeatability and from 1.16 to 3.15 for interday repeatability. The percentage of recovery values was between 94 to 115% (resins) and 80 to 103% (inflorescence extract). The cannabis industry is growing rapidly, and there is a need for reliable testing methods to ensure the safety and efficacy of cannabis products. In addition, current methods for cannabinoid analysis are often time-consuming and expensive, while the HPLC-UV method herein reported is a simple, rapid, accurate, and cost-effective alternative for the analysis of cannabinoids in cannabis resins, inflorescences, and medicinal oils. This method will be proposed to be included in the Cannabis sativa L. monograph of the Argentine Pharmacopoeia.

Plasma Cabozantinib Level Measurement in Patients with Renal Cell and Hepatocellular Carcinomas Using a Simple HPLC-UV Method Suitable for Clinical Application.

Cabozantinib, which is used to treat renal cell and hepatocellular carcinomas, is often associated with dose-dependent adverse events. Monitoring the levels of cabozantinib in the blood may maximize the therapeutic effect and prevent serious adverse events. In this study, we developed a high-performance liquid chromatography-ultraviolet (HPLC-UV) method of measuring plasma cabozantinib concentration. Human plasma samples (50 µL) were processed by simple deproteinization with acetonitrile, followed by chromatographic separation on a reversed-phase column with an isocratic mobile phase of 0.5% KH₂PO4 (pH 4.5) and acetonitrile (43:57, v/v) at a flow rate of 1.0 mL/min, with a 250 nm ultraviolet detector. The calibration curve was linear over the concentration range (0.05-5 µg/mL) with a coefficient of determination of 0.99999. The accuracy of the assay ranged from -4.35% to 0.98%, and recovery was >96.04%. The measurement time was 9 min. These findings confirm the effectiveness of this HPLC-UV method for cabozantinib quantification in human plasma, which is sufficiently simple for use for monitoring patients in clinical settings.

Development of a Simple Isocratic HPLC-UV Method for the Simultaneous Analysis of Repaglinide and Metformin Hydrochloride in Nanoemulsion Formulations and Commercial Tablets

Development of a Simple Isocratic HPLC-UV Method for the Simultaneous Analysis of Repaglinide and Metformin Hydrochloride in Nanoemulsion Formulations and Commercial Tablets

Holistic chemical characterization of Zaoren Anshen preparations based on HPLC fingerprint and multi-components quantification

Based on HPLC fingerprint analysis and multi-components quantification, a simple, sensible, and reliable HPLC-UV method, capable of qualitatively and quantitatively characterizing the main chemical markers of all medicinal herbs of Zaoren Anshen (ZRAS) prescription by one injection, was firstly developed. Then, three different dosage forms of ZRAS preparations, i.e., capsule, granule, and oral liquid, were analyzed and compared systematically, indicating the similarity and difference in chemical markers of the three ZRAS preparations. The strategy of combining HPLC chemical fingerprint and multi-components quantification in one injection was rather practical, powerful and useful in characterizing the main chemical markers of ZRAS preparations, which was a benefit for holistic quality evaluation and control of ZRAS preparations. Moreover, the present results provide sound scientific data for further studies on the effective constituents and quality control of ZRAS preparations.

Validated HPLC-UV method for determination of venlafaxine and its metabolite - o-desmethylvenlafaxine in human plasma

This paper describes a simple, rapid and cost-effective HPLC-UV method for the determination of venlafaxine and its active metabolite O-desmethylvenlafaxine in human plasma. Sample preparation is based on a liquid-liquid extraction procedure with a short extraction time. Prazosine hydrochloride was used as the internal standard. The HPLC separation was performed on a Supelcosil LC-CN column, using a mobile phase consisting acetonitrile : 25 mM phosphate buffer at pH 3,5 (15:85 v/v). The calibration curve is linear in the concentrations range of 25-1000 ng/mL, which is suitable for pharmacokinetic studies of venlafaxine hydrochloride following of dosing from 37.5 mg to 375.0 mg per day. The method was fully validated according to the international guidances,

Validation of Rapid and Simple HPLC-UV Method for Diflunisal Determination in Bulk Drug and Human Plasma

Abstract: Background: Transthyretin amyloidosis is a rare disease currently under wide investigation and many different therapeutic agents were developed for its control and treatment. In addition to the newly discovered drugs, it was also observed drug repurposing of some wellstudied therapeutic agents. Diflunisal was developed in 1971 as an anti-inflammatory and analgesic drug but showed good results when used as a kinetic stabilizer of the transthyretin protein. Objectives: Present study describes a high–performance liquid chromatography method for its determination in bulk drug and human plasma by UV detection. Materials and Methods: Isocratic elution of the mobile phase (consisting of 0.1% trifluoroacetic acid in water and acetonitrile in the ratio 42:58 v/v) at a flow rate 1.0 ml/min was set and the developed analytical procedure became fast and simple. Chromatographic determination was performed on a Purospher® RP – 18 column at room temperature and a UV detector set at 230 nm. Results: The developed method was validated for linearity in the range 0.5–125 μg/ml for the bulk drug and 0.48–120 μg/ml for plasma. Calibration curves were linear over the selected ranges with correlation coefficients (R2) greater than 0.999. The coefficients of variation for intra- and interassay were <2% for both methods – bulk drug and plasma determination. Conclusion: The developed effective and specific method can be applied in routine clinical practice for drug therapy monitoring. Keywords: Diflunisal, Bioanalysis, HPLC-UV, Validation.

An analytical quality by design approach towards a simple and novel HPLC-UV method for quantification of the antifibrotic peptide N-acetyl-seryl-aspartyl-lysyl-proline

N-acetyl-seryl-aspartyl-lysyl proline (Ac-SDKP) is a tetrapeptide possessing anti-fibrotic, angiogenic, anti-inflammatory, anti-apoptotic, and immunomodulatory properties. Currently, the main method to quantify the peptide is liquid chromatography-tandem mass spectrometry (LC-MS/MS) and enzyme-linked immunosorbent assay (ELISA), both of which are labour intensive and require expensive equipment and consumables. Furthermore, these techniques are generally utilised to detect very low or trace concentrations, such as in biological samples. The use of high concentrations of analyte might overload the extraction column or the separation column in LC-MS/MS or the ELISA plates, so the response could be a non-linear relationship at high analyte concentrations. Thus, they are not ideal for formulation development where detection of dose-equivalent concentrations is typically required. Therefore, a cost-effective, simple, and accurate quantification method for the peptide at a higher concentration needs to be developed. In this study, a simple and novel HPLC-UV method is proposed and validated using an Analytical Quality by Design (AQbD) approach. The method is first screened and optimised using chromatographic responses including capacity factor, resolution, tailing factor, and theoretical plate counts, fulfilling the International Council for Harmonisation (ICH) Q2 (R1) guidelines. The resultant optimised chromatography conditions utilised 10 mM phosphate buffer at pH 2.5 and acetonitrile as mobile phases, starting at 3% (v/v) acetonitrile and 97% (v/v) buffer and increasing to 9.7% (v/v) acetonitrile and 90.3% (v/v) buffer over 15 min at a flow rate of 1 mL/min at the column temperature of 25 °C. The injection volume is set at 10 μL and the VWD detector wavelength is 220 nm. The method established is suitable for detecting the peptide at a relatively high concentration, with a quantifiable range from 7.8 μg/mL to 2.0 mg/mL. In addition, the use of a relatively simple HPLC-UV approach could significantly reduce costs and allow easier access to quantify the peptide concentration. A limitation of this method is lower sensitivity compared with using LC-MS/MS and ELISA methods but running costs are lower and the methodology is simpler. The method is capable to quantify the peptide in various tested matrix solutions, with successful quantitation of the peptide in samples obtained from in vitro drug release study in PBS and from a chitosan-TPP nanogels formulation. Therefore, the method developed here offers a complementary approach to the existing quantification methods, quantifying this peptide at increased concentrations in simple to intermediately complex matrix solutions, such as HBSS, DMEM and FluoroBrite cell culture media.

Improved HPLC method for the determination of ribavirin concentration in red blood cells and its application in patients with COVID-19.

Ribavirin is a synthetic, broad‐spectrum antiviral drug. Ribavirin is recommended as an antiviral drug in the Interim Guidance for Diagnosis and Treatment (the seventh edition) of COVID‐19. The ribavirin levels in red blood cells may be closely related to both its efficacy and adverse drug reactions. In this study, a simple and fast HPLC–UV method was established to determine the concentrations of total ribavirin in the red blood cells of 13 patients with COVID‐19. Phosphorylated ribavirin was dephosphorylated by phosphatase incubation to obtain the total amount of ribavirin in red blood cells. The chromatographic column was an Atlantis C18. The recoveries were 85.45–89.05% at three levels. A good linear response was from 1 to 200 μg/ml, with a correlation coefficient of r 2 = 0.9991. The concentration of total ribavirin in the red blood cells of the patients ranged from 30.83 to 133.34 μg/ml. The same samples without phosphatase incubation ranged from 4.07 to 20.84 μg/ml. About 85% of ribavirin was phosphorylated in red blood cells. In addition, we observed changes in these patients' hematological parameters and found that the erythrocyte, hemoglobin and hematocrit declined to the lowest levels on the fifth day after discontinuation of ribavirin (p < 0.05).

Development of a Simple and Rapid HPLC-UV Method for Ultrasound-assisted Deep Eutectic Solvent Extraction optimization of Ferulic Acid and Antioxidant Activity from Ixora javanica Flowers

In this work, we developed an environmental friendly extraction method to enhance ferulic acid yields and antioxidant activity of I. javanica flowers using Ultrasound-assisted extraction (UAE) method and Deep Eutectic Solvents as green solvent. To optimize the extraction method, response surface method analysis using Box Behnken designs were conducted. The results showed that the optimum extraction conditions for ferulic acid yields, DPPH scavenging capacity, ABTS scavenging capacity and Ferric-Reducing Antioxidant Power featured an extraction time of 40 min, 25% water content, and solid-to-liquid ratio of 1/27 g/mL. In this study, a rapid and simple analytical method was also validated for the quantification of ferulic acid in I. javanica flower extract using LC-UV method. The developed method was carried out under following condition: isocratic mobile phase of acetonitrile/formic acid pH 2.55 (30:70), a flow rate of 0.8 ml/min, detection at 321 nm, and C-18 column (250 mm x 4.6 mm, 5 µm). The ferulic acid and antioxidant activity of the extract obtained from the developed were higher than ethanolic extract under same extraction condition. In summary, the results of this study can provide more effective and efficient extraction method by applying the green extraction principle and be recommended to provide new potential antioxidant raw material.

Simultaneous Determination of Prasugrel and its Process Related Impurities and Degradation Products in Bulk Drug Substances by RPLC-UV

Aims: This study aims to determine the quantitative prasugrel (PG) and its all possible process-related impurities. Background: To the best of our knowledge, very few analytical methods are available in the literature for monitoring process related impurities and degradation products of PG in bulk drug substance/ active pharmaceutical ingredient (API). Objective: The objective of this study is the separation of Prasugrel and its all possible process-related impurities viz., desacetyl prasugrel-tautomeric forms, intermediates including desacetyl impurity existing in its keto-enol form and positional tautomer impurities with degradation products. Method: A simple and robust HPLC-UV method having Zorbax XDB C18 column (15 cm x 4.6 mm) 3.5μm particle size column was used. Result: Prasugrel and its process related impurities were separated as well as analyzed in pharmaceutical samples' biological matrices. Conclusion: RP-LC method was developed for quantitative determination of PG and related substantial impurities were found to be highly specific, sensitive and precise. The major oxidative degradant was identified as PG desacetyl IMPs (keto-enol and positional tautomer) and hydroxyl IMP.

Study of phenolic composition of olive fruits: validation of a simple and fast HPLC-UV method

A high-performance liquid chromatography (HPLC) method endowed with a gradient elution and a UV detection system was established and validated for the determination of phenolic acids, phenolic alcohols, hydroxycinnamic acid derivatives, flavonoids, secoiridoids and lignans during olive (Olea europaea L.) fruit development (green, purple and black olives). Within the test range, the calibration curves exposed a good linear regression (R 2&gt;0.9995). Detection limits ranged between 0.63 and 13.43 mg/L for the detected phenolic compounds. The presented method yielded satisfactory repeatability in terms of retention times and average concentrations of phenolic compounds (RSD &lt; 0.3%). Verbascoside was established as the major phenolic compound in black olives. Oleuropein was established as the dominating phenolic compound in green olives, and its level decreased during maturation. Additionally, this research is the first to experimentally evidence that the flavone luteolin-7-rutinoside is the predominant flavonoid glucoside in black olives, showing the most significant variation with fruit development. The above results validate the method for an easy and fast determination of different classes of phenolic compounds present in olive fruits.

Development and validation of simple and sensitive HPLC-UV method for ethambutol hydrochloride detection following transdermal application.

A new high-performance liquid chromatographic method coupled with UV detection (HPLC-UV) to quantify ethambutol (ETH) post permeation studies following microneedle administration has been developed. This method involves the derivatization of ETH with phenethyl isocyanate (PEIC) at room temperature for 90 min. The separation of the derivative was performed using a C18 column that utilised a mobile phase consisting of 25 mM sodium dihydrogen phosphate buffer (with 1% v/v triethylamine, pH 3.0 adjusted using orthophosphoric acid) and methanol (25 : 75 v/v). The developed analytical method was validated according to the standards set by the International Council on Harmonization (ICH) guidelines. The method is linear for drug concentrations within the range of 0.39-12.5 μg mL-1 (R2 = 0.9999). The validated method was found to be specific, precise, and accurate. Moreover, the ETH derivative was found to be stable under specific storage conditions. In addition, a simple and straightforward extraction procedure for extracting and quantifying ETH from the skin was developed and evaluated. The extraction procedure displayed recovery rates that range from 101.77 ± 7.10% to 102.33 ± 8.69% indicating high extraction efficiency. The developed method was utilised in assessing the permeation of ETH across dermatomed neonatal porcine skin following microneedle application. Collectively, the simple and stable HPLC method developed in this study may be of great utility in screening formulations for ethambutol within a preclinical setting through in vitro permeation studies.

A simple HPLC-UV Method for Therapeutic Drug Monitoring of Linezolid in human Plasma in low-resourced settings

OBJECTIVE: A high-performance liquid chromatography method for the estimation of Linezolid in human plasma was developed and validated. METHODS: Samples (100µµL) were deproteinized with acetonitrile and analyzed using LiChrospher 100, RP18e column with PDA detection at 254 nm. The flow rate of the isocratic mobile phase comprising of 0.1% formic acid in 1000 ml of water and acetonitrile in the ratio of 60:40 (v/v) was set at 1.0 ml/min. RESULTS: The calibration curve ranged from 0.50 to 20.0 µg/ml and was linear. The recovery ranged from 96% to 101%. The accuracy ranged from 98 to 101% and intra- and inter-day relative standard deviation was &lt;4.58%. The method reliably eliminated interfering materials from plasma and R2 was 0.9973. The method described was applied to the determination of plasma LZD concentration in multi-drug-resistant tuberculosis patients who are treated with a dose of 600 mg LZD once daily. CONCLUSIONS: The developed method is suitable for determination of plasma LZD in routine care and considered feasible in less-resourced settings

Validated Simple HPLC-UV Method for Mycophenolic Acid (MPA) Monitoring in Human Plasma. Internal Standardization: Is It Necessary?

The aim of the work was to prepare a simple but reliable HPLC-UV method for the routine monitoring of mycophenolic acid (MPA). Sample preparation was based on plasma protein precipitation with acetonitrile. The isocratic separation of MPA and internal standard (IS) fenbufen was made on Supelcosil LC-CN column (150 × 4.6 mm, 5 µm) using a mobile phase: CH3CN:H2O:0.5M KH2PO4:H3PO4 (260:700:40:0.4, v/v). UV detection was set at 305 nm. The calibration covered the MPA concentration range: 0.1–40 µg/mL. The precision was satisfactory with RSD of 0.97–7.06% for intra-assay and of 1.92–5.15% for inter-assay. The inaccuracy was found between −5.72% and +2.96% (+15.40% at LLOQ) and between −8.82% and +5.31% (+19.00% at LLOQ) for intra- and inter-assay, respectively, fulfilling acceptance criteria. After a two-year period of successful application, the presented method has been retrospectively calibrated using the raw data disregarding the IS in the calculations. The validation and stability parameters were similar for both calculation methods. MPA concentrations were recalculated and compared in 1187 consecutive routine therapeutic drug monitoring (TDM) trough plasma samples from mycophenolate-treated patients. A high agreement (r2 = 0.9931, p < 0.0001) of the results was found. A Bland–Altman test revealed a mean bias of −0.011 μg/mL (95% CI: −0.017; −0.005) comprising −0.14% (95% Cl: −0.39; +0.11), whereas the Passing–Bablok regression was y = 0.986x + 0.014. The presented method can be recommended as an attractive analytical tool for medical (hospital) laboratories equipped with solely basic HPLC apparatus. The procedure can be further simplified by disapplying an internal standard while maintaining appropriate precision and accuracy of measurements.

High-performance liquid chromatographic detection of selected phenolic acidsin wine

The aim of this study was to develop a method for identification and quantification of phenolic acids in different wine samples. The simple reversed-phase HPLC-UV method for simultaneous determination of p-coumaric and ferulic acid was developed. The method was validated and working range, linearity, repeatability, accuracy, limit of quantitation LOQ and limit of detection LOD were determined. The linearity of the method was tested in concentration ranges 0.1-1 mg L-1 and 1-10 mg L-1. The correlation coefficients (r2) were greater than 0.996 and quality coefficients (QC) ≤ 5%. Detection limit for both compounds was 0.01 mg L-1. The method is precise (RSD