Reversed-phase C18 Column Research Articles

Combination of coagulant-assisted and SPE purification for the HPLC determination of lycorine and galanthamine in various foods

A simple high performance liquid chromatography (HPLC) method has been developed for the analysis of lycorine and galanthamine, typical alkaloids of narcissus, in food. Samples were extracted with sonication using water containing poly-aluminium chloride (PAC) and sodium hydroxide (NaOH) as a solvent followed by solid phase extraction (SPE) with hydrophilic-lipophilic balanced (HLB) cartridge. Elution of the analytes was achieved with 0.1 v/v% formic acid kept at 95 °C. Purification with PAC, which is normally used as a coagulant, was successfully combined with SPE, demonstrated the perfect strategy for the removal of interfering substances from the foods. Separation of the analytes was achieved on reversed phase C18 column. The analytes were monitored doubly at wavelengths of 240 and 290 nm for the detection. The method demonstrated excellent linearity with coefficients of determination (r(2)) higher than 0.999 over the range of 0.2–20 µg ml−1 for both lycorine and galanthamine. Limits of quantification (LOQs) of the method were in the range of 0.15–0.52 µg ml−1. Method performance was evaluated, demonstrated to have quantitative recoveries of 92.8–98.9% with satisfactory intra-day precision of lower than 3.7% and inter-day precision of lower than 5.0%. The method was also applied to a wide variety of foods with complex matrices, indicated the potential of practical use in plant poisoning caused by narcissus contaminated foods.

Development and validation of a HPLC-UV method for the simultaneous detection and quantification of paclitaxel and sulforaphane in lipid based self-microemulsifying formulation

Paclitaxel (PTX) and sulforaphane (SFN) are known anticancer molecules. Their activity was found to be potentiated when tested concurrently. Only recently, however, a novel SFN enabled PTX self-microemulsifying formulation (SMEDDS) was developed for their simultaneous delivery. This necessitated the development of an analytical method for the simultaneous detection and quantitation of PTX and SFN. In this study, a simple and sensitive isocratic high performance liquid chromatography-ultraviolet (HPLC-UV) analytical method was developed and validated per International Conference on Harmonization guidelines to satisfy this objective. Its application was demonstrated when quantifying the amount of PTX and SFN released from the SMEDDS in various dissolution media. The separation of the analytes was performed with the aid of a reversed phase C18 column at ambient temperature using a 60:40 mixture of acetonitrile and KH2PO4 buffer (pH5.0) as the mobile phase. PTX and SFN peaks were detected at 202nm with high resolution without interference from excipients. This method showed linearity within 2.5-100μg/mL range with r2>0.999. The limit of detection and lower limit of quantitation were 0.1638 and 0.4964μg/mL for PTX and 0.4419 and 1.3389μg/mL for SFN, respectively. A total of 98-101% of the injected samples was recovered with RSD of 0.06-0.68% indicating the suitability of the method for the simultaneous detection and quantitation of the molecules in dissolution media.

Chemometric optimization of an HPLC method for the simultaneous analysis of a multi component drug product by the help of central composite design

ABSTRACT In this study, a simple, accurate, precise and rapid HPLC method was developed for the simultaneous quantification of paracetamol (PAR), pseudoephedrine hydrochloride (PSE), dextromethorphan hydrobromide (DEX) and doxylamine succinate (DOX) along with colorants; allura red (AR) and brilliant blue (BB) and sweetener; saccharine (SAC) in a syrup sample. Three indepented variables considered and their levels in Central Composite Design in order to optimize chromatographic separation conditions were as follows: Phosphate buffer ratio in mobile phase in the first gradient elution (GR1; 67, 70, 75, 80 and 83%), phosphate buffer ratio in mobile phase in the second gradient elution (GR2; 27, 30, 35, 40 and 43%) and flow rate of the mobile phase (FR; 0.9, 1.0, 1.2, 1.4 and 1.5 mL/min). Critical resolution values between SAC, PAR and PSE along with between AR and BB were used as a response. Chromatographic separation was achieved using reversed phase C18 column (4.6 mm × 250 mm × 5 μm particle size) and the six analytes were detected with a diode array detector at 210 nm. As per the results of the optimization procedure, these optimum variables were found to be flow rate, 1.4 mL/min, phosphate buffer ratios in mobile phase in the first and second gradient elution, 75 and 40%, respectively. After optimization step, the developed method was validated in compliance with ICH guideline. Finally, the method was successfully applied to the simultaneous quantification of PAR, PSE, DEX, DOX, AR, BB and SAC in a commercial syrup sample.

Simultaneous detection of biogenic amines and aminobutyric acid isomers in high-protein forages

High performance liquid chromatography (HPLC) was applied for simultaneous determination of selected biogenic amines and non-proteinogenic amino acids in fermented forage, i.e. silage. The four most frequently occurring biogenic amines in silage and three isomers of aminobutyric acid, potentially impairing welfare and health of farm animals, were separated in a single analysis. Without time-consuming extraction and concentration procedures, these compounds were analysed as N-substituted phenylthiocarbamyl derivatives using a reversed-phase C18 column. The method is suitable for rapid determination of the extent of amino acid decomposition during fermentation in forage with high protein content.

Development and validation of a simple method for the extraction and quantification of crisaborole in skin layers.

Crisaborole is a boron compound recently approved by the US Food and Drug Administration as a 2% ointment for the treatment of mild to moderate atopic dermatitis. This work describes a simple method for the quantification of the drug in the skin layers at the end of in-vitro permeation experiments. Chromatographic separation was carried out on a reverse-phase C18 column using a mixture of trifluoroacetic acid 0.05%-acetonitrile (55:45, v/v) as mobile phase, pumped at 1 ml/min. Column temperature was 35°C and UV detection was performed at 250 nm. The method was linear in the range of concentration from 0.06 to 6 μg/ml (R2 = 1) and was selective, precise and accurate. Depending on the solvent used, the LOQ ranged from 0.014 to 0.030 μg/ml and the LOD from 0.005 to 0.010 μg/ml. The extraction from all the skin layers was quantitative. The developed method was successfully tested in an in-vitro permeation study, proving to be an effective tool in the development of new formulations containing crisaborole.

Optimization of an Ultrasound-Assisted Extraction for Simultaneous Determination of Antioxidants in Sesame with Response Surface Methodology.

Sesame is a nutritional agricultural product with medicinal properties. Accurate determination of micronutrients is important for the improvement of sesame quality and nutrition assessments. Our previous study showed that 10 antioxidants—d-homoproline, vitamin B2, coniferyl aldehyde, hesperidin, phloretin, N-acetyl-l-leucine, l-hyoscyamine, ferulic acid, 5-methoxypsoralen, and 8-methoxypsoralen—in sesame were potential characteristic nutrients in sesame. Herein, simultaneous detection of 10 different types of antioxidants was developed by using ultrasound-assisted extraction coupled with liquid chromatography-tandem mass spectrometry (UAE-LC-MS/MS) with the help of response surface methodology. The significant variables and levels were screened and optimized by combining the single factor experiment, Plackett–Burman test, and Box–Behnken design. The optimal conditions for extraction of target antioxidants in sesame were methanol solution of 75.0%, liquid-to-material ratio of 20:1 (mL/g), extraction temperature of 50 °C, extraction power of 410.0 W, extraction time of 65 min. The total yield of targets was 21.74 μg/g under the optimized conditions. The mobile phase used was 0.1% formic acid in acetonitrile and 0.1% formic acid in water, and the column was a Thermo Syncronis C18 reverse phase column (100 mm × 2.1 mm, 3 μm). All targets required only one injection and could be quickly separated and assayed within 7 min. The limits of detection and limits of quantification for these 10 nutritional compounds ranged from 0.01 to 0.11 µg/kg and from 0.04 to 0.34 µg/kg, respectively. The validation results indicated that the method had reasonable linearity (R2 ≥ 0.9990), good recoveries (71.1%–118.3%), satisfactory intra-day precision (≤9.6%) and inter-day precision (≤12.9%), and negligible matrix effects (≤13.8%). This simultaneous quantification method was accurate, fast, and robust for the assessment of sesame nutrition.

Overcoming the Charged Ion Competition in Triple Quad Chemical Ionization Ion Source for Estimation of Celecoxib in Biological Matrix and its Application to Bioequivalence Study.

The purpose of the study was to develop and validate a sensitive, selective and reproducible method for the estimation of Celecoxib in human plasma. During the development, ion competition phenomenon observed in electrospray ionization ion source of liquid chromatography mass spectrometer system and in order to overcome it, different approaches applied. The Celecoxib and Celecoxib D7 in plasma were extracted by liquid-liquid extraction and separated in less than 4.0 min on a reverse phase C18 column, using isocratic elution with a binary mobile phase in the ratio of 70:30 v/v (Acetonitrile: 0.1% Ammonia Solution). Mass spectrometer was used as detector to quantitate the analytes in positive ion mode, using Atmospheric Pressure Chemical Ionization mode. Since the method was developed to perform the pharmacokinetic end point study for United States and Europe market, the method was fully validated, complying Food and Drug Administration, European Medicines Agency guideline and recommendations of American Association of Pharmaceutical Scientists white papers.

HPLC-DAD method for simultaneous determination of natural polyphenols

A simple and reliable high-performance liquid chromatography with diode-array detection (HPLC-DAD) method was developed for simultaneous determination of 9 natural substances common in plants: three major catechins ((-) - epicatechin gallate, (-) - catechin, (-) - epigallocatechin), four major flavonoids (rutin, quercetin, myricetin, kaempferol), gallic and vanillic acid. The optimized method was carried out for 40 minutes with detection wavelengths of 278 and 368 nm, gradient elution system on a C18 reversed-phase column. The developed system was evaluated for several validation characteristics, as system suitability, specificity, linearity, limit of detections (LODs) and limit of quantifications (LOQs). The newly established HPLC method was proved to be specific, sensitive, linear and precise. The received results showed good chromatographic separation and assumed that the described method was validated. The HPLC-DAD method can be a useful tool for the quantitative and qualitative evaluation of the selected polyphenol compounds and can be improved in the future for the examination of the polyphenols of interests in individual herbal infusions.

Chemical Constituents Isolated from Rhododendron ungernii with Antioxidant Profile

Background:Plant in Rhododendron genus that contains phenolic compounds has been used in traditional medicine and revealed considerable biological activities.Objective:Isolation and identification of antioxidant natural products from Rhododendron ungernii.Methods:Rhododendron ungernii Trautv. flowers were collected and dried in shade. The dried flowers were extracted with methanol for 3 days. The solvent was removed by reduced pressure to yield the extract which was subjected to column chromatography (Sephadex LH-20, C18 reversed phase column) to isolate catechin-7-O-glucoside (1), quercetin-3-O-β-galactoside (2), quercetin-3-O- β-xyloside (3), farrerol (4), myricetin (5), and quercetin (6). The structures of isolated compounds were elucidated by spectroscopic methods such as 1D-NMR, 2D-NMR, and LC-TOF/MS. DPPH scavenging effect, ABTS+ scavenging activity, and reducing power (FRAP) were performed for antioxidant assays of isolated natural compounds.Results:Isolated flavonoids displayed the outstanding antioxidant activities. Catechin-7-O-glucoside (1) and quercetin-3-O-β-galactoside (2) (IC50, 3.66 µg/mL) had the most DPPH• scavenging effect among the compounds. The highest ABTS•+ scavenging activity (IC50, 1.41 µg/mL) and reducing power effect (6.05 mmol TE/g comp) were observed for myricetin (5).Conclusion:R. ungernii extract and isolated compounds could be a promising antioxidant for food and pharmaceutical industries.

Liquid Chromatography/Mass Spectrometry Method for the Simultaneous Determination of Atorvastatin and Telmisartan in Human Plasma

A simple liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) method has been developed and validated for the simultaneous determination of Atorvastatin (ATO) and telmisartan (TEL) in human plasma. After liquid-liquid extraction step, the analytes and internal standard, rosuvastatin (ROS) were separated using an isocratic mobile phase consisting of 10 mM ammonium acetate (pH=4)-methanol (20:80, v/v) on a reverse-phase C18 column. Detection was carried out on mass spectrometer by using electrospray ionization (ESI) source using their respective [M+H] + ions, m/z 482 for ROS, m/z 515 for TEL, and m/z 559 for ATO. For both drugs, the method was linear in the range of 1-35 ng/mL. Acceptable precision and accuracy were obtained for concentrations in the calibration range.

Olaparib Quantification in Human Plasma for Clinical Purposes using High-Performance Liquid Chromatography with UV detector

A simple, sensitive and accurate HPLC-UV detection method for the determination of olaparib in human plasma samples was developed. Separation was successfully achieved using an YMC C18 reverse phase column (250 × 4.6 mm i.d.; 5 μm) under isocratic mobile phase conditions consisting of methanol-0.1 % orthophosphoric acid (60:40, v/v) and ultra-violet detection was achieved at 220 nm. Extraction of the target compounds was completed using liquid-liquid extraction technique. No endogenous substances were found to interfere with the peaks of drug and internal standard. Good linearity (r2 > 0.99) was achieved for the concentration range of 100-5000 ng/mL and detection limit was 20 ng/mL. The Intra and Inter-day precision and accuracy were found to be within acceptable limits (<15 %). The overall recovery for olaparib was found to be 50.44 %. Stability data revealed that the drug was stable in plasma under various test conditions. Matrix effects were not observed. The present method was effectively implemented to determine the concentration of olaparib in human plasma.

Determination of Salicylic Acid in Raw Milk by Ultra Performance Liquid Chromatography

A method for the detection of salicylic acid in raw milk was set up. The sample was extracted with acetonitrile and determined by ultra performance liquid chromatography, and separated on a ZORBAX Eclipse Plus C18 reversed-phase column, using gradient elution, with a mobile phase of phosphoric acid water and methanolic acid, and a DAD detection. The linear range of salicylic acid was 0.5~20 μg/mL, the correlation coefficient squared (R2) was more than 0.995, the detection limit was 0.5 mg/kg, the quantification limit was 1.25 mg/kg, the average recovery rate of the three different levels were between 88.0% to 118.9%, and the relative standard deviation was not more than 10%. The method had the characteristics of simple operation, good reproducibility, high sensitivity, small impurity interference and can be used for the detection of salicylic acid in raw milk.

МЕТОДИКА КОНТРОЛЯ СОДЕРЖАНИЯ СИНТЕТИЧЕСКИХ КРАСИТЕЛЕЙ В ПИЩЕВОЙ ПРОДУКЦИИ

Разработана и валидирована методика одновременного определения 17 синтетических красителей (СК) (Е 102, Е 104, Е 110, Е 121, Е 122, Е 123, Е 124, Е 127, Е 128, Е 129, Е 131, Е 132, Е 133, Е 142, Е 143, Е 151, Е 155) в различных группах пищевой продукции, включая молочную продукцию и продукты с высоким содержанием белка. Методика характеризуется простотой, надежностью, высокой чувствительностью и может быть легко применима в аккредитованных лабораториях. Процедура основана на экстракции красителей из пищевой продукции водно-метанольным раствором аммиака, центрифугировании, декантировании, доведении рН экстракта до 7 и количественном определении методом высокоэффективной жидкостной хроматографии с диодно-матричным детектированием. Для очистки экстрактов от липидов использовали гексан, молочные белки осаждали с помощью реактивов Карреза I, II. Хроматографическое разделение СК осуществлялось на обращенно-фазной колонке С18 (Zorbax XDB, 150 х 4,6 мм, 5,0 мкм) в режиме градиентного элюирования. Использовали подвижную фазу состава: A полутора процентный раствор ацетата аммония, В метанол : ацетонитрил (80 : 20). Предел количественного определения СК составил от 1 до 10 мг/кг. Максимальная расширенная неопределенность полученных результатов не превысила 23 процентов для каждого синтетического красителя. An analytical procedure for simultaneous determination of 17 synthetic dyes (i.e. E 102, E 104, E 110, E 121, E 122, E 123, E 124, E 127, E 128, E 129, E 131, E 132, E 133, E 142, E 143, E 151, and E 155) in different kinds of food products, including dairy food and high-protein food products, has been developed and validated. The procedure is characterized by simplicity, reliability, high sensitivity and can be easily applied in accredited laboratories. The workflow runs starting from the extraction of dyes from food products with an aqueous-methanol solution of ammonia followed by centrifugation, decantation, adjusting the extract pH value to 7 and subsequent quantitative determination using high performance liquid chromatography with diode array detection. Hexane is used to purify extracts from lipids; milk proteins are precipitated using Karrez I, II reagents. Chromatographic separation of synthetic dyes is carried out on a C18 reverse phase column (Zorbax XDB, 150 x 4.6 mm, 5.0 micrometers) in gradient elution mode. The mobile phase is as follows: A 1.5 percent solution of ammonium acetate, B methanol: acetonitrile (80 : 20). The quantitative limit for the synthetic dyes varies in the range from 1 to 10 mg/kg. The maximum expanded uncertainty of the results does not exceed 23 percent for each synthetic dye.

Secreted Metabolome of Human Macrophages Exposed to Methamphetamine.

Macrophages comprise a major component of the human innate immune system that is involved in maintaining homeostasis and responding to infections or other insults. Besides cytokines and chemokines, macrophages presumably influence the surrounding environment by secreting various types of metabolites. Characterization of secreted metabolites under normal and pathological conditions is critical for understanding the complex innate immune system. To investigate the secreted metabolome, we developed a novel workflow consisting of one Reverse Phase (RP) C18 column linked in tandem with a Cogent cholesterol-modified RP C18. This system was used to compare the secreted metabolomes of human monocyte-derived macrophages (hMDM) under normal conditions to those exposed to methamphetamine (Meth). This new experimental approach allowed us to measure 92 metabolites, identify 11 of them as differentially expressed, separate and identify three hydroxymethamphetamine (OHMA) isomers, and identify a new, yet unknown metabolite with a m/z of 192. This study is the first of its kind to address the secreted metabolomic response of hMDM to an insult by Meth. Besides the discovery of novel metabolites secreted by macrophages, we provide a novel methodology to investigate metabolomic profiling.

Spectrometric analyses of larotaxel and larotaxel liposomes quantification by high performance liquid chromatography

Larotaxel is a new chemical structure drug, which has not been marketed worldwide. Accordingly, the standard identification and quantification methods for larotaxel remain unclear. The spectrometric analyses were performed for verifying weight molecular formula, molecular weight and chemical structure of larotaxel. Besides, a quantification method was developed for measuring larotaxel in the liposomes. The molecular formula, molecular weight and chemical structure of larotaxel were studied by using mass spectrometry (MS), infra-red (IR), nuclear magnetic resonance (NMR) and ultraviolet-visible (UV-vis) spectrometric techniques. The absorption wavelength of larotaxel was investigated by UV-vis spectrophotometry full-wavelength scanning. Besides, a quantification method was developed by high performance liquid chromatography (HPLC), and then validated by measuring the encapsulation efficacy of larotaxel liposomes. The four spectral characteristics of larotaxel were revealed and the corresponding standard spectra were defined. It was confirmed that larotaxel had the structure of tricyclic diterpenoids, with the molecular formula of C45H53NO14, the molecular weight of 831.900 1, and the maximum absorption wavelength of 230 nm. The quantitative method of larotaxel was established by using HPLC with a reversed phase C18 column (5 μm, 250 mm×4.6 mm), a mobile phase of acetonitrile-water (75:25, volume/volume), and a detection wavelength of 230 nm. The validation study exhibited that the established HPLC method was stable, and had a high recovery and precision in the quantitative measurement of larotaxel in liposomes. In addition, a new kind of larotaxel liposomes was also successfully prepared. The particle size of the liposomes was about 105 nm, with an even size distribution. And the encapsulation efficiency of larotaxel in the liposomes was above 80%. The present study offers reference standard spectra of larotaxel, including MS, IR, NMR, and UV-vis, and confirms the molecular formula, molecular weight and chemical structure of larotaxel. Besides, the study develops a rapid HPLC method for quality control of larotaxel liposomes.

Quantitative monitoring of a panel of stress-induced biomarkers in human plasma by liquid chromatography-tandem mass spectrometry: an application in a comparative study between depressive patients and healthy subjects.

Using a metabolomic approach, we have found that stress can induce oxidative damage by disturbing the creatine/phosphocreatine shuttle system and purinergic pathway, leading to an excessive membrane breakdown. To further validate our findings and to monitor the biological impact of stress in research of clinical psychiatry, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed to simultaneously determine a panel of biomarkers comprising choline, creatine, purinergic metabolites, neurosteroids, lysophosphatidylcholines, and phosphatidylethanolamines in human plasma. After optimization of the extraction protocol, all the 15 analytes plus 4 internal standards with distinct polarities were extracted into an organic phase using methyl tert-butyl ether/methanol (1:1, v/v). A reversed-phase C8 column under gradient elution consisting of aqueous phase A of 5mM ammonium acetate buffer solution containing 0.1% formic acid and organic phase B of acetonitrile/2-propanol (3:7, v/v) was utilized for separation. Four sequential periods under positive or negative ion mode were combined for the determination of analytes with specific multiple reaction monitoring transitions. For all analytes, this method exhibited good linearity with coefficients of determination (R2) higher than 0.99. The lower limit of quantification (LLOQ) values ranged from 0.05 to 80.0ng/mL. Recovery between 70.5 and 97.3% was obtained by spiking standards to plasma samples stripped by powdered activated carbon. The intra- and inter-assay relative standard deviations (RSDs) of the analyses varied between 2.0 and 13.3%. The mean accuracy ranged from 90.6 to 109.0%. The matrix effect ranged from 91.2 to 107.3% with variations less than 9.0%. Stability under different conditions was tested, with mean recoveries varying between 90.4 and 109.7%. Finally, the established method was successfully applied to analyze the plasma samples from a small cohort of 30 patients with major depressive disorder and 30 matched healthy controls. Graphical abstract.

Mzabimycins A and B, novel intracellular angucycline antibiotics produced by Streptomyces sp. PAL114 in synthetic medium containing L-tryptophan

In our previous studies, the production of four bioactive molecules by Streptomyces sp. PAL114 in complex ISP2 broth medium has been described. Three of these molecules belong to the angucycline family. In this study, two novel antibiotics belonging to the same family were produced by strain PAL114 on M2 synthetic medium containing L-tryptophan as precursor. These antibiotics, named mzabimycins A and B, were intracellular and produced only in the presence of L-tryptophan. After four days of culturing PAL114 in the M2 medium, the bioactive compounds were extracted from mycelium with methanol and then analyzed by HPLC on reverse phase C18 column. Two active purplish blue fractions were purified. The chemical structures of these molecules were determined on the basis of spectroscopic and spectrometric analyses (1H and 13C NMR, and mass spectra). They were identified to be novel angucycline derivative antibiotics. The pure molecules showed activity against some pathogenic Gram-positive bacteria which have multiple antibiotic resistance, such as Staphylococcus aureus MRSA 639c and Listeria monocytogenes ATCC 13932.

Development and Validation of an UHPLC Method for Ostarine Determination in Dietary Supplements

Abstract Objective: The purpose of this study was to develop a low-cost, yet sensitive and precise UHPLC method for the quantitative determination of ostarine from dietary supplements (DS) for athletes. The analytical performance of the method was verified on a DS legally acquired from a specialized website for athletes. The uniformity of mass and content of the ostarine DS was also verified. Methods: For the quantitative determination of ostarine a UHPLC method was developed and validated. The separation was performed using a reversed-phase C18 column, using a mixture of 75% methanol: 25% formic acid 0.1% in isocratic elution, at a flow rate of 0.5 ml/min. The uniformity of mass and content of DS was performed following the methodology described in the European Pharmacopoeia 7th Edition. Results: The validated method was specific and linear on the concentration range of 1-25 µg/ml and was precise and accurate at all concentration levels, according to the official guidelines for validating analytical methods. An average mass of 510 mg content was obtained for the ostarine capsules, with an RSD of 2.41%. Regarding the uniformity of the content, an average of 4.65 mg ostarine/capsule was obtained with an RSD of 1.05%. Conclusions: The developed UHPLC method was suitable, rapid, sensitive and allowed quantitative determination of active substance content in a DS with ostarine (92.91% ostarine/capsule from 5 mg ostarine/capsule declared by the manufacturer).

Fast and Sensitive Quantification of Δ9-Tetrahydrocannabinol and Its Main Oxidative Metabolites by Liquid Chromatography/Tandem Mass Spectrometry.

Introduction: Few animal studies have evaluated the pharmacological effects of Δ9-tetrahydrocannabinol (THC) in relation to its pharmacokinetic properties. Understanding this relationship is essential, however, if comparisons are to be drawn across conditions—such as sex, age, and route of administration—which are associated with variations in the absorption, metabolism, and distribution of THC. As a first step toward addressing this gap, in this report, we describe a rapid, sensitive, and accurate method for the quantification of THC and its main oxidative metabolites, and apply it to representative rodent tissues.Materials and Methods: The sample workup procedure consisted of two steps: bulk protein precipitation with cold acetonitrile (ACN) followed by phospholipid removal by elution through Captiva-Enhanced Matrix Removal cartridges. The liquid chromatography/tandem mass spectrometry (LC/MS-MS) protocol utilized a commercially available C18 reversed-phase column and a simple methanol/water gradient system. The new method was validated following Food and Drug Administration (FDA) guidelines, and was applied to the quantification of THC and its main oxidative metabolites—11-hydroxy-Δ9-tetrahydrocannabinol (11-OH-THC) and 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (11-COOH-THC)—in plasma and brain of mice treated with a single intraperitoneal dose of THC (10 mg/kg).Results: ACN precipitation and column elution effectively depleted matrix constituents—most notably choline-containing phospholipids—which are known to interfere with THC analysis, with average recovery values of >85% for plasma and >80% for brain. The LC conditions yielded baseline separation of all analytes in a total run time of 7 min (including re-equilibration). The 10–point calibration curves showed excellent linearity (R2>0.99) over a wide range of concentrations (1–1000 pmol/100 μL). Lowest limit of quantification was 2 pmol/100 μL for all analytes, and lowest limits of detection were 0.5 pmol/100 μL for THC and 11-OH-THC, and 1 pmol/100 μL for 11-COOH-THC. Intraday and interday accuracy and precision values were within the FDA-recommended range (±15% of nominal concentration). An application of the method to adult male mice is presented.Conclusions: We present a fast and sensitive method for the analysis of THC, which should facilitate studies aimed at linking the pharmacokinetics and pharmacodynamics of this compound in animal models.

Otimização e validação intralaboratorial de método analítico por CLAE/UV para identificação e quantificação de p-fenilenodiamina em tinturas de hena para cabelos e sobrancelhas

Introduction: p-Phenylenediamine (PPD), aromatic sensitizing amine, has been added to henna powder to modify its natural color to black, increasing its fixation time, a practice that is prohibited in eyelash and eyebrows dyes. Objective: The objective of this study was to optimize and validate, at an intra-laboratory level, an analytical method by HPLC/UV for identification and quantification of PPD in hen dyes for hair and eyebrows. Method: In the method, C8 reverse phase column, mobile phase 1% triethanolamine (pH 8.4) andacetonitrile (99: 1, v/v), detection 280nm, injection volume: 10uL, flow 1.0 mL/min, column temperature 32°C, run time 10 min, linearity 5-45 μg/mL (n = 5) with correlation coefficient 0.9982, were used. For Cochran test (homoscedasticity): 0.3350 and Critical (0.3934) with 99% confidence. Limits of detection 1.17 μg/mL and quantification 3.54 μg/mL. The coefficient of variation of repeatability was 0.12% and the intermediate precision by F-test yielded p value of 0.283 with 95% of confidence. Accuracy results comprised acceptance criteria of 90%-107%. Results: Of the 19 analyzed samples, 14 presented PPD content between 1.74 and 3.65% w/w, in disagreement with Legislation. Conclusions: The proposed method can contribute to monitoring of quality and safety of use of these products.