The major processes for introducing polycyclic aromatic hydrocarbons (PAHs) in food are smoking and grilling of different products. But in addition, PAHs can permeate in the food chain due to their high lipophilicity and ability to be accumulated in specific tissue, through contaminated animal feed. Further, when some parts of these animals are marketed as food, the accumulated PAHs can go to the human organism. Some of them are classified as highly toxic, carcinogenic and mutagenic for animal and human organisms so they are under consideration of International and European legislation. This work reports development and validation of simple and fast GC/MS method for 16 PAHs determination. Comparison of two methods for sample preparation in pork meat matrix standard extraction/saponification procedure and modified QuEChERS method is also done. In addition, this paper report the calibration step of instrument and a recovery study for 16 PAHs in model pork meat, using modified QuEChERS procedure for sample pretreatment. The calibration step with accessible and suitable for use in real laboratory conditions internal standard (chrysene D12) is done in the range 10–100 ppb using toluene as solvent. The obtained results show very good linearity (R2 = 0.99 to 1.00). For the recovery study six model samples were spiked with 16 PAHs and they all are subjected to QuEChERS procedure. The recovery is calculated and the obtained data (71–120%) is in a good correlation with requirements of international legislation. Finally, LOD values for all 16 investigated compounds of modified GC/MS method and for the instrument were determined.
In the last few years, the use of surfactants as mobile phase additives in reversed phase liquid chromatography (RPLC) has been steadily developing and improving. Surfactants modify the polarity of the stationary phase which in turn decreases the amount of organic solvent required for elution of the analytes rendering the methodologies linked to them greener and more eco-friendly. Brij-35 is a fatty alcohol ethoxylates non ionic surfactant, which is less widely used as mobile phase additive. Brij-35 can decrease stationary phase polarity while remaining neutral. In this research, Brij-35 was studied in the separation and determination of marketed antihypertensive combination therapy composed of triamterene (TRM) and xipamide (XIP). TRM and XIP are diuretics used for treatment of essential hypertension and associated edema conditions. Chromatographic separation was achieved on RP-C18 column (Kinetix®, 5 µm, 15 cm × 4.6 mm) at flow rate 1 mL min−1 and UV-detection at 254 nm. Isocratic elution was performed using mobile phase composed of 0.1 M Brij-35: methanol (MeOH) (60:40, v/v). The analytes were well separated and quantified within linearity ranges of 5–50 µg mL−1 for both drugs in short retention time (2.6 and 5.3 min. for TRM and XIP, respectively). Since claiming greenness is not enough, Green Analytical Procedure Index (GAPI) was used to demonstrate the superiority of the proposed method over the previously reported methods. GAPI is a new metric for evaluation of the ecological impact of analytical procedures. The proposed method was validated according to ICH guidelines and applied successfully for simultaneous determination of the drugs in their co-formulated tablets.
A simple HPLC technique has been utilized for rapid and sensitive quantitative analysis of two mixtures of drugs that are used during pregnancy and lactation. Drugs of the first mixture are used to manage gastrointestinal tract illness that are common during early stages of pregnancy, while pharmaceutical agents of the second mixture are administered over the counter as galactagogues or to overcome postpartum depression. Mixture I includes famotidine (FMT), ranitidine (RNT), nizatidine (NZT), and pantoprazole (PNT), which were separated on a C18 column using a mobile phase composed of methanol: 0.02 M sodium dihydrogen phosphate (60:40, v/v) of pH 6.9, adopting UV detection at 240 nm at a flow rate of 1 mL/min. Mixture II on the other hand, consists of domperidone (DOM), metoclopramide (MET), and sulpiride (SUL). These drugs were eluted using the same column and flow rate as those in mixture I, using a mobile phase consisting of acetonitrile: 0.075 M sodium dihydrogen phosphate (30:70, v/v) of pH 6 adopting a detection wavelength 270 nm. Two optimization protocols were utilized to optimize the chromatographic separation conditions, namely one factor at a time (OFAT) and design of experiments (DOE) where face centered cube response surface experimental design was chosen for this investigation. Comparison of the results obtained from both protocols reveals the accordance between them.
Full validation procedure under guidance of United States Pharmacopoeia (USP) was applied to the proposed methods which enabled their application to separate the drugs of both mixtures in spiked rat whole blood samples and in vivo analysis of rat heart blood.
Palmatine is a compound with good water solubility extracted from Coptis chinensis, Fibraurea recisa Pierre, Cortex Phellodendri Chinensis. Palmatine has good antibacterial activity and mainly used for the treatment of bacterial dysentery, gynecological inflammation, surgical infection, and conjunctivitis. It has anti-diabetic, anti-oxidant, and cognitive-enhancing activities. In this study, we used UPLC-MS/MS to determinate palmatine in rat plasma, and investigated its pharmacokinetics. Coptisine was utilized as an internal standard (IS), and acetonitrile precipitation method was used to process the plasma samples. Chromatographic separation was achieved using a UPLC BEH C18 column using mobile phase of acetonitrile- 0.1% formic acid with gradient elution. Electrospray ionization (ESI) tandem mass spectrometry in multiple reaction monitoring (MRM) mode with positive ionization was applied. The results indicated that within the range of 1–500 ng/mL, linearity of palmatine in rat plasma was acceptable (r > 0.995), and the lower limit of quantification (LLOQ) was 1 ng/mL. Intra-day and inter-day precision RSD of palmatine in rat plasma were less than 14%. Accuracy range was between 93.7 and 107.1%, and matrix effect was between 101.6 and 109.4%. The method was successfully applied in the pharmacokinetics of palmatine in rats after oral and intravenous administration. The absolute bioavailability of the palmatine was 15.5% in rats.
Vicia faba, also known as “bakla” in Turkey, is a species of Fabaceae family that is widely grown in Africa and Asia. It is rich in levodopa, a medicinal substance used to treat Parkinson's disease. Levodopa produced by chemical synthesis is expensive and causes various side effects. Therefore, it is recommended to use natural levodopa sources to prevent possible side effects. A Central Composite Design technique has been used in this study to optimize levodopa extraction from Vicia faba. First, a single factor analysis examined 3 variables such as extraction temperature, extraction time, and concentration of acetic acid. The purpose of this study was to assess the effects of variables chosen on levodopa's extraction performance. By using variance and regression analyses, a second-order regression equation was determined as a predicted model. The value of R2 is 0.9882, which shows that the equation fits well. The best conditions are as follows: a temperature of 59.85 °C, an extraction time of 18.74 min, and an acetic acid content of 0.28%. Under optimum conditions, the maximum levodopa yield calculated from the predicted module was 4.53%. Extraction efficiency was determined as 4.54% experimentally under optimum conditions. A good relationship has been found between the experimental result and the predicted value.
Cortisol and cortisone are 2 important glucocorticoids produced in the human hypothalamus–pituitary–adrenal (HPA) axis that respond to stress. An analytical method to determinate cortisol and cortisone in serum and saliva using high-performance liquid chromatography–tandem mass spectrometry following a supported liquid extraction (SLE) was developed. Serum and saliva samples of 0.2 mL were extracted by SLE three times using 0.4 mL of methyl tert-butyl ether each time. The chromatographic separation was obtained on an Agilent Poroshell column using a 0.01% formic acid buffer and acetonitrile (60:40, v/v) as the solvent with a flow rate of 0.3 mL/min. Optimized quantitative mass transitions for cortisol, cortisone, and cortisone d-4 were 363.2/121.0 (m/z), 361.2/163.1 (m/z), and 367.1/270.7 (m/z), respectively. The method validation was achieved according to regulatory guidance. The lower limit of quantification (LLOQ) in serum were 2 ng/mL for cortisol and 1 ng/mL for cortisone, and the LLOQ in saliva were 0.1 ng/mL for cortisol and 0.2 ng/mL for cortisone. The developed method showed convenient and efficient extraction, a lower LLOQ, and a short running time. Modest correlations between serum and saliva cortisol and cortisone concentrations were found. The method was successfully applied in assessing the HPA condition of patients with depressive disorders.
The aim of this study was to develop a novel reversed-phase high-performance liquid chromatography (RP-HPLC) method for efficient separation of ivabradine and its 11 impurities. Similar polarity of impurities in the sample mixture made method optimization challenging and accomplishable only when different chemometric tools, such as principal component analysis (PCA), Box–Behnken design (BBD), and desirability function as a multicriteria approach, were employed. The presence of 3 positional isomers (impurities III, V, and VI), keto–enol tautomerism of impurity VII, and diastereoisomers of impurity X made separation of this complex mixture even more challenging. Chromatographic retention parameters obtained with the mobile phase consisting of 30 mM phosphate buffer and acetonitrile (80:20, v/v) on four different RP-HPLC columns at varying pH values (3.0, 4.0, and 5.0) were subjected to the PCA analysis to select the column with the most appropriate selectivity. Then the column temperature, pH of the aqueous component of mobile phase, phosphate buffer molarity and the organic solvent content in the mobile phase were estimated employing BBD. Valid and reliable mathematical models towards resolution of twelve critical peak pairs were obtained. After determination of the desirability making criteria for all responses, desirability functions were established and used in optimization. The proposed optimal chromatographic conditions included the Zorbax Eclipse Plus C18 chromatographic column (100 × 4.6 mm, 3.5 μm), the column temperature of 34 °C, the mobile phase flow rate of 1.6 mL min−1 and the UV detection at 220 nm. The mobile phase consisted of the 28 mM phosphate buffer at pH 6.0 and acetonitrile (85:15, v/v). Separation of one pair of positional isomers was not achieved, so methanol was added to the organic part of mobile phase in small increments with the optimal ratio of methanol to acetonitrile 59:41, v/v. The overall organic component of the mobile phase also increased to 18%, accelerating the chromatographic analysis.
We developed and validated an assay for determination of glyphosate (GLYP) and glufosinate (GLUF) in human serum. Serum samples were extracted by using a MonoSpin® TiO column and analyzed by liquid chromatography–tandem mass spectrometry (LC–MS/MS). MonoSpin® TiO tends to specifically bind to phosphate groups. The assay was linear over a concentration range of 1–250 μg/mL. The recoveries for the 2 compounds were 1.6%–2.3%. The intra- and inter-day variations were <15%. Precision and accuracy were 5.6%–12.7% and 97.0%–103.9%, respectively. The validated method was applied to quantify the GLYP and GLUF content in the serum of GLYP and GLUF-poisoned patients. In conclusion, the method was successfully applied for accurate determination of GLYP and GLUF in serum obtained from patients with GLYP and GLUF poisoning.
Coffee is one of the most widely consumed beverages in the world. It contains many bioactive compounds, including chlorogenic acid which possesses various biological properties. In this study, in order to determine concentration of chlorogenic acid in green coffee, a reverse-phase rapid resolution liquid chromatography (RP-RRLC) method with diode-array detection (DAD) was developed. Successful separation was achieved on a Poroshell 120 EC-C18 (50 mm × 3 mm; 2.7 μm) column using acetonitrile–water with 1% phosphoric acid (10:90, v/v) as a mobile phase, at a flow rate of 1 mL/min, and with UV detection at 325 nm. The identification was made with comparison of the retention time of pure analytical standard with the retention time of chlorogenic acid in the analyzed samples. The developed method was validated using the following parameters: linearity, sensitivity, selectivity, precision, and accuracy. Excellent linearity over the range 12.33–143.50 μg/mL was achieved with R2 values greater than 0.99. The intra-day precision was validated with the %RSD values, which confirmed that the method for determination of chlorogenic acid was repeatable. The mean recovery rate of the method ranged between 97.87% and 106.67% with %RSD values lower than 1%. The limit of detection and limit of quantification values under the used chromatographic conditions were 0.29 and 0.96 pg, respectively. This method was successfully employed for quantitative determination of chlorogenic acid in green coffee samples.
The advent of disposable micro-columns will be a hope of workers of chromatography-related laboratories. A very critical and important requirement is the formation of affordable inlet frits. Welding a metal screen to a column inlet is not recommended because of the risk of damage to stationary phase. In this study, the Tollens probe (silver mirror reaction) was adopted to make affordable frits. Silver is reduced on the particle surface and in an empty space among the particles, forming a solid silver network structure at the column inlet area by injecting the reaction solution into the packed column at a depth of one third (10 cm) of the packed bed (0.5 mm × 300 mm). The silver cement structure was successfully formed, and the silver cement frit endured mobile phase flow well when C18 modified ground silica monolith particles were used to make the packed bed. The formation of the silver cement frit was not successful when the stationary phase based on conventional spherical silica particles was used. Negligible reduction of chromatographic performance by the silver cemented frit was observed. This study serves as the first step toward realization of disposable micro-columns.