Mobile Phase Of Water Research Articles

QbD-Driven Development and Validation of a Bioanalytical LC–MS Method for Quantification of Paliperidone in Human Plasma

This paper discusses how a Quality by Design (QbD) strategy was used to develop and test an HPLC-MS bioanalytical method for detecting plasma Paliperidone concentration. A C18 column and an isocratic mobile phase of organic solvents and water were improved for chromatographic separation. To optimise method performance, Box-Behnken design was used to study column temperature, mobile phase composition, and flow rate. The research used a logical QbD methodology to build an optimised HPLC-MS technology that meets US-FDA standards. Validation studies evaluated selectivity, sensitivity, carryover effects, matrix factor determination, linearity, accuracy and precision testing, recovery, dilution veracity, ruggedness, stability, and reinjection reproducibility, with supporting documentation. The validation studies showed the method's suitability and met acceptance requirements. The QbD framework was successfully used to build and validate an HPLC-MS bioanalytical technique for Paliperidone measurement in human plasma. This method improves chromatographic performance and Paliperidone quantification in clinical and pharmacokinetic studies.

Development of simultaneous quantitative analytical method for three active components of Korean mint (Agastache rugosa (Fisch. & C.A.Mey.) Kuntze) extract in human plasma using ultra-high-performance liquid chromatography–tandem mass spectrometry

Agastache rugosa contains phenolic compounds and flavonoids, and has been extensively used as a traditional herbal medicine. The major components in Agastache rugosa extract (ARE) are rosmarinic acid, tilianin, and acacetin, for which several analytical techniques have been reported. However, these substances have yet to be simultaneously quantified in human plasma. In this study, we aimed to simultaneously determine the three active components of ARE in human plasma by developing a reliable quantitative analytical method using ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS). Chromatographic separation of the plasma samples was achieved using an ACQUITY UPLC® BEH C18 column with a gradient mobile phase of water and acetonitrile containing 0.1 % formic acid. Mass spectrometric detection was performed using a triple quadrupole tandem mass spectrometer in negative electrospray ionization (ESI−) and multiple reaction monitoring (MRM) modes. The developed quantitative method was validated for the three active components. All three analytes exhibited a linear response over the ranges of 0.5–50 ng/mL for rosmarinic acid, 0.1–20 ng/mL for acacetin, and 0.5–20 ng/mL for tilianin with a weighting factor of 1/x (where x is the concentration). At three quality control (QC) concentration levels (low, medium, and high), including the lower limit of quantitation (LLOQ), acceptable accuracy (±15 %) was achieved in the intra- and interday validations. The concentration of rosmarinic acid was highest in plasma. Tilianin and acacetin appeared and were eliminated earlier in the plasma than rosmarinic acid. This study provides a successfully validated method that can be used in further clinical applications of Agastache rugosa extracts.

Stability indicating RP-HPLC method development and validation for estimation of Crisaborole in topical dosage form

A simple, specific, accurate, precise , rapid, robust and selective stability indicating reverse phase high performance liquid chromatography (RP-HPLC) method has been developed for assay and related substances and validated for quantification of Crisaborole with its excipients in its topical dosage form. Forced degradation study is done to determine stability of the product. In mobile phase Water: Methanol (30: 70 v/v), the maximum of Crisaborole was measured to be 241nm. The technique is highly sensitive, with linearity ranging from 5 to 25g/ml (regression equation: y = 316606x - 427583; r2 = 0.9992). For verifying and testing this approach, several parameters according to ICH recommendations and USP are used. The detection and quantitation limits were determined to be 0.1094 and 0.3316 g ml-1, respectively. The results revealed that the process is accurate, specific, and repeatable (RSD 2%), as well as easy, inexpensive, and less time consuming, and suitable for determining Crisaborole in topical dosage form. The method have been robust under various variation with flow rate, detection wavelength and column oven temperature. The drug was exposed to stress conditions. Method resolves all degraded product as compared to Crisaborole. Developed method can be used routinely for estimation of drug Crisaborole with its excipient methyl paraben in dosage form and stability sample.

Eco-friendly liquid chromatography method for the quantification of ibrutinib in a pharmaceutical dosage form.

The objective of this study was to quantitatively determine Bruton's tyrosine kinase inhibitor ibrutinib in its capsule dosage form and assess the homogeneity of the dosage form using green chromatography. The chromatographic method using gradient elution mode was optimized and validated in accordance with the International Council for Harmonization guidelines. The analysis was conducted on a Zodiac C18 column (75 × 4.6 mm and 3.5 μm) using a mobile phase consisting of pH 5.5 potassium phosphate buffer (mobile phase A) and 90% ethanol in milli-Q water (mobile phase B), with a flow rate set at 0.6 mL/min. Based on the validation data, the accuracy results fell within the range of 99.1%-100.6%. The relative standard deviation (% RSD) from precision for both the assays and the uniformity of dosage by content uniformity were determined to be 0.82 and 1.16, respectively. The correlation coefficient obtained from the linearity experiment was 0.999, indicating a strong linear relationship. The greenness of the developed method was assessed using various tools, including the National Environmental Methods Index (NEMI) pictogram, Modified NEMI, Analytical Eco-score calculation, Green Analytical Procedure Index (GAPI) pictogram, Analytical GREEnness (AGREE), and AGREE preparation (AGREEprep). The obtained greenness profile suggests that the optimized LC method is an excellent greener method, supported by the analytical eco-score of 86.

A novel UHPLC-MS/MS method for trace level identification and quantification of genotoxic impurity 2-(2-chloroethoxy) ethanol in quetiapine fumarate

A selective and sensitive new reversed-phase method was developed and validated by using Waters Acquity UHPLC system coupled with Waters Micromass Quattro PremierXE (MS/MS) to identify and quantify the genotoxic impurity 2-(2-chloroethoxy) ethanol at trace level (∼2.0 ppm) in quetiapine fumarate. The method used positive ion electrospray ionization with a multiple reaction monitoring detection mode using ACE3 C18(100 mm × 4.6 mm × 3.0 µm) column. For analysis, an isocratic program was developed using 0.01 M ammonium acetate in Milli-Q water (mobile phase A) and methanol (mobile phase B) in 20:80v/v ratio. Elution of 2-(2-chloroethoxy) ethanol was monitored using triple quadrupole mass spectrometer with an injection volume of 10 μl, a column oven temperature of 40 °C, an auto-sampler temperature of 15 °C, and flow rate of 0.5 mL/min. The retention time of 2-(2-chloroethoxy) ethanol was observed at 2.38 min. The LOQ and LOD were determined at concentrations of 0.235 ppm and 0.070 ppm respectively. The correlation coefficient R 2 was 0.9983 and the percent recoveries of method range from 98.1% to 114.0%. The method was successfully validated according to International Council for Harmonization requirements (ICH Q2(R1) and ICH M7(R1)) for genotoxic impurities. The method was sensitive, selective, accurate and precise and therefore can be used for identification and quantification of genotoxic impurity 2-(2-chloroethoxy) ethanol in quetiapine fumarate.

A rapid method for detecting bronopol in fresh fish, shrimp, crab, and shellfish samples using liquid chromatography-tandem mass spectrometry

Fish farming plays a vital role in providing food, nutrition, and employment globally. However, this industry faces security challenges, necessitating the use of fungicides and preservatives, such as bronopol, to increase product yields. Bronopol (2‑bromo-2-nitropropan-1,3-diol; CAS:52–51–7) is widely used in various fields, including food production, cosmetics, and, more recently, aquaculture. Currently, there is a limited number of techniques available for detecting bronopol in aquaculture products. This is primarily due to bronopol's instability, susceptibility to degradation, and tendency to form precipitates that pose challenges in extraction from aquaculture products. For this issue, this study presents a comprehensive method for detecting bronopol content in aquaculture tissues using liquid chromatography-tandem mass spectrometry (LC‒MS/MS). The methodology was optimized, involving extraction with Cu-Zn precipitant, cleanup using a small HLB column, separation on a T3 column, and gradient elution with water and acetonitrile mobile phases. The quantitative approach was employed without the use of an internal standard, following the external standard method. The spiked recoveries at 3 fortification levels (0.1, 0.2, and 1 mg/kg) ranged from 87.1 % to 108.1 % with relative standard deviations RSD ≤ 9.0 %. By applying this method to fresh fish, shrimp, crab, and shellfish samples from a local supermarket, no residues of bronopol were detected, ensuring the reliability of the results. The simplicity, rapidity, and high sensitivity of the method make it a suitable alternative to conventional techniques for bronopol detection. Moreover, the successful validation of the method's recovery and precision supports its potential application in monitoring and preventing the misuse of bronopol in aquaculture, thereby safeguarding aquaculture product quality and protecting public health.

An extensive screening method for the identification and quantitation of ecdysteroids in equine urine and plasma using liquid chromatography coupled with mass spectrometry.

Recently, there has been a report suggesting that ecdysteroids can enhance sports performance, making them relevant substances in doping control. Hence, it is imperative to examine the analytical characteristics of ecdysteroids in biological samples to identify their misuse in competitive sports. To assess the doping of ecdysteroids such as ecdysone, ecdysterone, ponasterone A, turkesterone, and ajugasterone C, a fast and sensitive extraction and detection method was developed, optimized, and validated using equine urine and plasma. Different extraction techniques, namely, solid-phase extraction, liquid-liquid extraction, and dilute-and-inject, were explored to detect ecdysteroids from equine urine and plasma. The most suitable method of detection was solid-phase extraction using ABS Elut-NEXUS, while liquid-liquid extraction and dilute-and-inject methods encountered difficulties due to the high polarity of ecdysteroids and the presence of significant matrix interferences. Mass spectrometric parameters are optimized on both the Q Exactive high-resolution mass spectrometer and the TSQ Altis triple quadrupole mass spectrometer. However, the study indicated that the triple quadrupole mass spectrometer exhibited improved limit of detection when analyzing samples. To achieve optimal separation of the analytes under investigation from the matrix interferences, various liquid chromatography columns were compared. The Selectra PFPP LC column with a mobile phase consisting of 0.2% formic acid in water (mobile phase A) and acetonitrile (mobile phase B) at a flow rate of 0.5 mL/min demonstrated superior performance. The findings of this study will significantly contribute to the accurate identification of ecdysteroids, facilitating the investigation of their illicit use in horse racing.

Quality Control and Authentication of Black Betel Leaf Extract (Piper acre Blume) from East Kalimantan as an Antimicrobial Agent Using a Combination of High-Performance Liquid Chromatography and Chemometric Fourier Transform Infrared.

Black betel leaf from East Kalimantan contains various secondary metabolites such as alkaloid saponins, flavonoids, and tannins. A compound, piperenamide A, which has antimicrobial activity, is also found in black betel leaf. This study aims to identify and authenticate the compound piperenamide A found in black betel leaf extract in other types of betel plant using HPLC and FTIR-chemometrics. The extraction method used was maceration with 70% ethanol solvent. Determination of piperenamide A content in black betel leaf extract was via HPLC column C18, with a maximum wavelength of 259 nm and a mobile phase of water:acetonitrile at a flow rate of 1 mL/minute. From the results, piperenamide A was only found in black betel (Piper acre) and not in Piper betel and Piper crocatum. Piperenamide A levels obtained were 4.03, 6.84, 5.35, 13.85, and 2.15%, respectively, in the samples studied. The combination of FTIR spectra with chemometric methods such as PCA and PLS-DA was used to distinguish the three types of betel. Discriminant analysis can classify black betel (Piper acre), Piper betel, and Piper crocatum according to its type. These methods can be used for identification and authentication of black betel.

Analytical Method Development and Validation for Estimation of Lapatinib in Formulation by RP-HPLC with Stability Indicating

Reverse-phase high-performance liquid chromatography (RP-HPLC) approach for Lapatinib in pharmaceutical dosage form development and validation. Reverse phase chromatography is easy, practical, and superior in terms of effectiveness, stability, and repeatability. Lapatinib was chosen to be separated using the C18 column, a 250 x 4.6 mm column with 5.0µm particle packing. As lapatinib was exhibiting good peak morphologies and a large amount of resolution, it was created with a mobile phase of water, methanol, and trifluroacetic acid (30:70:0.1) v/v. The analytes were detected at 262nm using a UV detector while the mobile phase was flowing at 1.1 ml/min. Specificity, linearity, accuracy, precision, robustness, limit of detection, and limit of quantitation were used to develop the method and validate it.The method demonstrated a dynamic linear response at 25-75 µg/ml and was shown to be linear with a correlation coefficient (r2) of above 0.999 and limits of detection and quantitation (LOD and LOQ) of 0.45 and 1.35µg, respectively. Degradation of the sample was used to establish the stability indicating RP-HPLC procedures, which were then compared to standards. The relative standard deviation as a percentage was also less than 2%, demonstrating the suggested method's high level of precision.

Simultaneous LC-MS/MS quantification of SGLT2 inhibitors and antipyrine in medium and tissue from human ex vivo placenta perfusions

A liquid chromatography – tandem mass spectrometry (LC-MS/MS) method has been developed to simultaneously measure four sodium glucose co-transporter 2 (SGLT2) inhibitors and the transfer marker antipyrine (ANTI) in perfusion medium and placental tissue collected from ex vivo human placental perfusions. The four SGLT2 inhibitors were empagliflozin (EMPA), dapagliflozin (DAPA), ertugliflozin (ERTU) and canagliflozin (CANA). Chromatographic separation was achieved on an Uptisphere® C18 reversed phase column (50 mm × 4.6 mm × 5 µm) within 2.85 min, using a gradient elution with 10 mM ammonium formate in water (mobile phase A) and acetonitrile (mobile phase B) both with 0.1% formic acid. Analysis of ammonium adduct ions was performed on an AB SCIEX 6500+ triple quadrupole mass spectrometer using positive electrospray ionisation and scheduled multiple reaction monitoring (sMRM). The transitions were m/z 468.00 → 355.20 (EMPA), m/z 426.00 → 167.20 (DAPA), m/z 437.10 → 206.90 (ERTU), m/z 462.00 → 249.00 (CANA) and m/z 189.20 → 55.90 (ANTI).The method was validated according to the European Medicines Agency guidelines and was proven to be selective, linear within a concentration range of 1–1000 µg/L (DAPA, CANA, ANTI) and 1–500 µg/L (EMPA, ERTU), accurate, precise and free of carry-over, instabilities, recovery and matrix effect issues.This newly developed method is suitable to analyse perfusion medium and placenta tissue samples collected during ex vivo human placenta perfusions. It thereby enables quantification of transport across the placental barrier of the SGLT2 inhibitors EMPA, DAPA, ERTU and CANA as well as the transfer marker ANTI.

Bioanalytical method development and Validation for determination of efonidipine hydrochloride ethanolate in rat plasma by high-performance liquid chromatography: applications for pharmacokinetic study

Efonidipine hydrochloride ethanolate (EFE), a dihydropyridine calcium channel blocker, is used for the treatment of hypertension. To find the EFE in pre-clinical samples (rat plasma), an HPLC method was devised for the current experiment. The biological samples were prepared using the methanolic extraction procedure to eliminate solvent effects. Using a C18 column, a pH-adjusted mobile phase of methanol and water (90:10, v/v) at a flow rate of 1 mL/min, and UV detection at 252 nm were utilized for chromatographic separation. The retention time of EFE was found to be 5.2 min. The developed bioanalytical method was validated with different validation parameters. The validation parameter results are within the acceptable range of the International Council for Harmonization guidelines. The method was successfully employed to evaluate the pharmacokinetic properties in Wistar albino rats following a single oral administration of 10 mg/kg of EFE. Therefore, the simple method would make it possible to detect EFE quickly and affordably from biological material.

Preparation of polyvinyl pyrrolidone stationary phase and its application in separation and analysis of flavonoids

AbstractPolyvinylpyrrolidone (PVP) is known as a polymer with special adsorption properties for polyphenolic compounds. In this paper, chemical‐bonded stationary phase (PVP@Silica) was prepared and applied to the separation of flavonoids. System constants determined by linear solvation energy relationships with methanol–water mixtures as the mobile show that the column can provide stronger hydrogen bonding and π–π interaction than C18 column. Under reversed‐phase conditions, traditional flavonoids have much longer retention on PVP@Silica than on C18 column with methanol–water mobile phase due to multimodal retention mechanism, including hydrogen bonding and π–π interaction. In addition, a U‐shaped retention curve was observed in acetonitrile–water mobile phase because of the enhanced hydrogen bonding under the high proportion of acetonitrile. The selectivity to polyhydroxy structure gives the stationary phase unique separation ability for flavonoids. The separation orthogonality was further investigated by a sample set containing 33 flavonoids with different substitution structures. The (RP‐PVP@Silica)‐(RP‐C18) system exhibited 60% orthogonality metric (OM) for these flavonoids with methanol–water mobile phase. A high OM of 63.5% was achieved in (RP‐PVP@Silica)‐(HILIC‐PVP@Silica) system. Finally, PVP@Silica was applied to the purification of total flavonoids in Ginkgo biloba extract (GBE). The offline (RP‐PVP@Silica)‐(RP‐C18) two‐dimensional separation system was used for the analysis of flavonoids in GBE.

Development of Efficient Analytical method for the multicomponent analysis of Pravastatin Sodium and Nebivolol Hydrochloride in Bulk Drug by RP-HPLC

Cardiovascular diseases are managed through multi drug regimens. For better patient compliance and cost effectiveness, desired therapeutic efficacy is achieved through combination of more than two drugs in single dosage form. Nebivolol Hydrochloride is β1 receptor blocker and Pravastatin sodium exhibits its pharmacological action through inhibition of cholesterol synthesis. Both drugs are therapeutically compatible and can be administered in single dose formulation. A review of the literature found that there was no effective strategy for the selected drug combination. As a result, an attempt was made to establish a simple, efficient, sensitive, specific, and rapid method for simultaneously estimating Pravastatin sodium and Nebivolol hydrochloride in bulk drugs using RP-HPLC.Column C-18 (Shim-pack) 250 x 4.6 mm, particle size 5 m, mobile phase acetonitrile and water in the ratio 20:80 at 257 nm were found to be the optimal chromatographic conditions. Pravastatin sodium and Nebivolol hydrochloride had retention times of 2.092 and 3.623 minutes, respectively, with average percentage recovery of 99.38% and 99.54%, respectively.The proposed technique was found to be in accordance with the guidelines of the International Conference on Harmonization (ICH).

Characteristics, Isolation Methods, and Biological Properties of Aucubin.

Aucubin is an iridoid glycoside widely spread in the families Cornaceae, Garryaceae, Orobanchaceae, Globulariaceae, Eucommiaceae, Scrophulariaceae, Plantaginaceae, and Rubiaceae. This review is intended to provide data on the physicochemical characteristics, isolation methods, and biological activities of aucubin and its producing plants. Aucubin is unstable and can be deglycosylated into its aglycone, aucubigenin. Various chromatographic methods (column chromatography, vacuum liquid chromatography, medium pressure liquid chromatography, and high-performance liquid chromatography) have been used together to isolate aucubin, mainly with the stationary phase C-18 and the mobile phase water-methanol solution made in gradients. In vitro and in vivo studies reveal that aucubin has a wide range of activities, including anti-inflammatory, antioxidant, anxiolytic and antidepressant, antidiabetic, antifibrotic, antimicrobial, anticancer, antihyperlipidemic, gastroprotective, cardioprotective, hepatoprotective, retinoprotective, neuroprotective, osteoprotective, and renoprotective. Even though aucubin has been extensively investigated, further research in humans is urgently needed primarily to substantiate the clinical evidence. Moreover, extensive studies on its drug delivery systems will help maximize efficacy and minimize side effects.

A facile approach to undecylenic acid-functionalized stationary phases for per aqueous liquid chromatography

Green chromatography techniques using low-toxic mobile phase are getting increasingly attention in recent years. The core is developing stationary phases that possess adequate retention and separation under the mobile phase of high content water. Using thiol-ene click chemistry, an undecylenic acid-bonded silica stationary phase (UAS) was prepared in a facile manner. Elemental analysis (EA), solid-state 13C NMR spectroscopy and Fourier transform infrared spectrometry (FT-IR) confirmed the successful preparation of UAS. The synthesized UAS was employed for per aqueous liquid chromatography (PALC), which uses little organic solvent during separation. Due to the hydrophilic carboxy, thioether group and hydrophobic alkyl chains of the UAS, various categories of compounds (including nucleobases, nucleosides, organic acids and basic compounds) with different properties can achieve enhanced separation under the mobile phase of high content water compared with commercial C18 and silica stationary phases. Overall, our present UAS stationary phase shows excellent separation ability toward highly polar compounds and meets the requirements of green chromatography.

Development and validation of a simple HPLC-MS/MS method for the quantification of methylmalonic acid without a derivatization step in human serum.

A simple and rapid HPLC-MS/MS analytical method was developed and validated for the determination of methylmalonic acid without a derivatization step in human serum. Serum samples with a volume of 200 μL were pre-treated in a simple way based on ultrafiltration using a VIVASPIN 500 ultrafiltration column. Chromatographic separation was achieved on a Luna Omega C18 column with a PS C18 precolumn guard by a gradient elution using 0.1% (v/v) formic acid in water (component A of the mobile phase) and 0.5% (v/v) formic acid in acetonitrile (component B of the mobile phase) with flow rate 0.2 ml.min-1 . The total run time of the analysis was 4.5 minutes. Negative electrospray ionization and multiple reaction monitoring mode were used. The lower limit of detection (LLOD) and lower limit of quantification (LLOQ) for methylmalonic acid were determined to be 13.6 nmol. L-1 and 42.3 nmol. L-1 , respectively. The developed method allows the quantification of methylmalonic acid in a wide linear range of 42.3-4230 nmol. L-1 with a correlation coefficient of 0.9991.

Investigation of a Quantitative Bioanalytical HPLC Method for the Assessment of Famotidine in Pre-clinical Samples: Application for Pharmacokinetic Studies

Famotidine (FAM), a histamine H2 receptor antagonist, is effective against peptic ulcers and Zollinger Ellison syndrome. In the current investigation, a HPLC approach was developed to detect the Famotidine (FAM) in pre-clinical samples (rat plasma). To get rid of any chromatographic solvent effects, methanolic extraction method was used to prepare biological samples. A C18 column was used for chromatographic separation, with a pH-adjusted to 8 as mobile phase of water, methanol, and acetonitrile (70: 20: 10, v/v/v) at a flow rate of 1 mL/min and UV detection at 264 nm. The retention times of FAM was found to be 5.7 min. The technique was linear in the range of 50 to 1400 ng/mL in plasma concentration with R2 of 0.9985. The newly proposed method’s respective limits for quantification and detection were found to be 151.45 ± 6.47 ng/mL and 49.97 ± 2.14 ng/mL. The average recovery rates were discovered to range from 98.06 to 103.56%. After numerous freeze-thaw cycles of the treated plasma samples, analyte was stable and showed no sign of significant deterioration. After a single oral dose of 10 mg/kg of FAM, the approach was effectively used to assess the pharmacokinetic characteristics in Wistar albino rats. Consequently, the straightforward technique would enable quick with affordable detection of the FAM from biological samples.

A validated LC–MS/MS method for clinical pharmacokinetics and presumptive phase II metabolic pathways following oral administration of Andrographis paniculata extract

Andrographis paniculata, a medicinal plant in Thailand national list of essential medicines, has been proposed for treatment of patients with mild to moderate coronavirus disease 2019. This study aims to develop a highly selective and sensitive liquid chromatography triple quadrupole tandem mass spectrometry method for quantitative determination of major diterpenoids in plasma and urine with application in pharmacokinetics. Chromatographic separation was performed on C18 column using a gradient mobile phase of water and acetonitrile. Mass spectrometry was analyzed using multiple reaction monitoring with negative ionization mode. This validated analytical method was very sensitive, less time consuming in analysis, and allowed the reliability and reproducibility on its application. The clinical pharmacokinetics was evaluated after single oral administration of A. paniculata extract (calculated as 60 mg of andrographolide). The disposition kinetics demonstrated that major diterpenoids could enter into systemic circulation, but they are mostly biotransformed (phase II) into conjugated glucuronide and sulfate metabolites. These metabolites are predominantly found in plasma and then extremely eliminated, in part through urinary excretion. The successful application of this analytical method supports its suitable uses in further clinical benefits after oral administration of A. paniculata.

Simple HPLC-UV Method for Piperacillin/Tazobactam Assay in Human Plasma

Piperacillin (Pip)/tazobactam (Taz) is a broad-spectrum antimicrobial agent that has been commonly used in the intensive care unit for severe and life-threatening infections. Recent evidence suggests that therapeutic drug monitoring (TDM) for Pip could be beneficial in clinical practice to facilitate dose optimization and increase the odds of treatment success. The aim was to develop and validate a sensitive and simple high-performance liquid chromatography (HPLC) method for the simultaneous quantification of Pip and Taz in human plasma. Samples (0.3 mL) were deproteinized with acetonitrile. The supernatant was evaporated and then reconstituted and injected into the HPLC. The chromatographic analysis was carried out by using the C18 column and gradient elution with the acetonitrile:water mobile phase mixture with 0.1% trifluoracetic acid at a flow rate of 0.8 mL/min using a UV detector at 218 nm. The method had acceptable linearity (r2 > 0.99) over the concentration ranges of 0.5-400 μg/mL and 1-100 μg/mL for Pip and Taz, respectively. The method demonstrated acceptable inter- and intra-day precision and accuracy within ±20% with adequate stability results. The developed method is sensitive and simple and utilizes simple sample preparation and elution steps, making it suitable and practical for Pip/Taz TDM.

Pipetting sample preparation with water eluent followed by water mobile phase HPLC analysis for residual monitoring of melamine in milk

The author introduces a small-scale, time shortening, economical sample preparation with water eluent followed by an isocratic water phase HPLC system for quantifying melamine in cow’s milk. Sample preparation is achieved by homogenization using a handheld ultrasonic-homogenizer with water followed by MonoTipⓇC18 pipette tip contains silica monolith bonded with octadecyl group with water eluent. For determination and identification of analyte, the HPLC uses an analytical C18 column, an isocratic 100% water, and a photo-diode array detector. The procedure, performed under a 100% water conditions, uses no organic solvents and poisonous reagents at all and is, therefore, harmless to both humans and the environment. The method validation data were well within the international method acceptance criteria. The total analytical time and quantitation limit were < 6 min/sample and 0.24 μg/mL, respectively. The present technique may be proposed as an international harmonized analytical method for routine residue monitoring for melamine in milk.