Limit Of Quantification Concentrations Research Articles

Analysis of Metaldehyde in Animal Whole Blood and Serum by Gas Chromatography-Mass Spectrometry.

Metaldehyde, a widely used molluscicide, is the third cause of intoxication by pesticides in domestic animals in Europe. Most mammalian species are susceptible, and its exposure may lead to death within a few hours. While metaldehyde intoxication diagnosis is in most cases presumptive, based on the symptomatology or from "postmortem" analysis, few analytical methods are currently available for live animals. The aim of this work was to describe a fast analytical method for the specific and quantitative determination of metaldehyde in animal whole blood and serum at concentrations of toxicological significance. A liquid-liquid extraction with chloroform and gas chromatography-mass spectrometry quantification are proposed. The method limit of quantification (LOQ) was 0.04 µg/mL in serum and whole blood. The method was linear in the range from 0.04 to 200 µg/mL. The recovery was between 93% and 102% for LOQ, low, medium and high spike concentrations. Intra- and inter-assay relative standard deviation was <12% in all spike concentrations in both serum and whole blood, apart from one of the experiments at LOQ in whole blood, which accounted for 17.7%. The method was applied to real intoxication cases, and the concentration found in positive samples was between 29 and 69 µg/mL. The proposed method provides high sensitivity, accuracy and precision and can be used to assist in the diagnosis of metaldehyde poisoning.

Development and validation of RP-HPLC method for quantification of trace levels of topical corticosteroids in ambiphilic cream

AbstractCorticosteroids are anti-inflammatory and immunosuppressant drugs. Topical corticosteroids formulations (ointments, creams, gels) are used in the treatment of different types of dermatitis and urticaria. Considering their therapeutic and whitening effects, they are frequently used for counterfeiting of cosmetic products. Corticosteroids can cause different local and systemic side effects. HPLC method is often chosen for their analysis, because it is selective, sensitive, precise, simple and fast.The aim of this study was optimization and validation of RP-HPLC method with UV detection for determination of trace levels of corticosteroids in ambiphilic creams. This method is used for qualitative and quantitative analysis of evaluated corticosteroids.Mometasone furoate, hydrocortisone acetate, fluocinonide, fluocinolone acetonide, betamethasone, betamethasone dipropionate and triamcinolone acetonide were evaluated. Separation was performed on Inertsil® ODS-3V 250 × 4.6 mm, 5 μm chromatographic column. Mobile phase was mixture of acetonitrile and water 50:50 (v/v) with gradient elution and flow rate 1 mL min−1. Column temperature was held on 40 °C and UV detection was performed at 240 nm.Selectivity, linearity, accuracy, precision and limit of quantification (LOQ) were evaluated. Method is selective because ambiphilic cream base peaks and corticosteroids peaks were not overlapping. Linearity was confirmed since correlation coefficient was 1 for all compounds. Accuracy and precision were evaluated for hydrocortisone acetate and betamethasone dipropionate. Determined Recovery values were in range of 70–130%. Both RSD values (21.46% and 9.59%) were lower than 30%. Method is highly sensitive since LOQ concentrations were in ng mL−1 range.All evaluated parameters of validation were in accordance with regulatory requirements. Validated RP-HPLC method can be used for qualitative and quantitative analysis of selected corticosteroids in ambiphilic creams.

Validation of Chromatographic Methods: Determining the Limit of Quantification in Practice

Key issues related to determining the limit of quantification (LOQ) in practice are considered. Requirements to the value of LOQ and its precision and accuracy are formulated. It is shown that the use of a calibration curve for LOQ determination may result in erroneous conclusions for impurity determination methods. Answers to a number of questions relevant to practice are given. The precision of the LOQ value should always be checked if the LOQ is determined from a calibration curve. A typical requirement for LOQ precision is RSD ≤ 5.0%. An RSD ≤ 10% is acceptable in exceptional cases of a very low LOQ concentration. For this, the validation documentation should visually confirm the acceptability of the LOQ value by a chromatogram of a solution of the determined substance with the LOQ concentration.

Colorimetric Quantification Methods for Peracetic Acid together with Hydrogen Peroxide for Water Disinfection Process Control.

Peracetic acid (PAA) water solutions is applied for disinfection of industry systems, food products and non-potable water. Commercially available peracetic acid is always supplied mixed with hydrogen peroxide (H2O2). H2O2 degrade slower than the peracetic acid which creates a need to quantify both peroxides separately to gauge the disinfection power of the solution and the residuals. Two combinations of colorimetric reactions are presented that allows simultaneous quantification at the mg·L−1 level used in disinfection liquids and water disinfection. The first dichromic reaction use titanium oxide oxalate (TiO-Ox) which only react with H2O2 followed by addition of N,N-diethyl-p-phenylenediamine with iodide (DPD/I−) and the concentrations are read by simultaneously measuring the absorbance at 400 and 515 nm. Limit of quantification (LOQ) and maximal concentration determined was 4.6 µg·L−1 and 2.5 mg·L−1 for PAA and 9.1 µg·L−1 and 5 mg·L−1 for H2O2. The two color reactions didn’t interfere with each other when the reagent addition was consecutive. Another combination of colorimetric reaction also used where TiO-Ox was used to first measure H2O2 at 400 nm, before addition of 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS)) and reading the absorbance at 405 nm. ABTS changes the absorbance at 405 nm necessitating the two measurements be done separately. LOQ and maximal concentration determined using ABTS colorimetric assay was 42.5 µg·L−1 and 30 mg·L−1 for PAA and for titanium oxide oxalate colorimetric assay was 12.7 µg·L−1 and 75 mg·L−1 for H2O2. Both methods tested satisfactory in typical water samples (Tap, sea, lake, and biological treated sewage) spiked with peracetic acid and H2O2, separately.

A Single-Column Gas Chromatography Method for Quantifying Toxic Alcohols.

Rapid identification and quantification of toxic alcohols and ethylene glycol is imperative for appropriate treatment. Clinical laboratories frequently rely on direct injection gas chromatography (GC) methods, but these methods require inlet maintenance and multiple GC systems. To overcome these challenges, we developed a single-column headspace GC method for both toxic alcohols and glycols that streamlines patient sample analysis for toxic alcohol ingestion. Optimal parameters for nonderivatized (volatile) and derivatized (glycol) plasma samples were determined using a 7890 A headspace sampler, an Agilent 7697 A GC system, a DB-200 column, and a flame ionization detector. Limit of Quantification (LoQ), linearity, imprecision, carry-over, method comparison, and interference studies were performed using quality control materials and prepared plasma samples. Our volatile method is linear to 3000 mg/L (ethanol) with LoQ concentrations below 20 mg/L (ethanol). The glycol method is linear to 2000 mg/L (ethylene glycol) with LoQ concentrations below 40 mg/L (ethylene glycol). Total assay impression ranged from 1.7% for ethanol to 13.3% for propylene glycol. Both methods were free of sample carryover and compared favorably with a similar clinical method at an outside laboratory. Propionic acid, an accumulating metabolite in methylmalonic acidemia that interferes with ethylene glycol identification by a different method, did not interfere with the ethylene glycol method reported here. Our single-column headspace GC method provides reliable, robust, and rapid identification and quantification of commonly encountered toxic alcohols. Clinical laboratories relying on direct injection Gas Chromatography (GC) for toxic alcohol analysis face challenges including frequent inlet maintenance, sample carryover, or the need for separate GC systems for volatile and glycol analysis. We summarize our development and optimization of two headspace GC methods for nonderivatized (volatile) and derivatized (glycol) plasma samples that use a single DB-200 analytical column. These methods are comparable to other GC methods, not prone to sample carryover, eliminate the need for multiple GC systems or columns, and are readily applicable to other laboratories that provide toxic alcohol analysis.

Mixed monolayer decorated SPR sensing surface for thrombin detection

The development of surface plasmon resonance (SPR) based immunosensor for thrombin detection was aimed. For this purpose, 3,3′ Dithiodipropionic acid di (N-hydroxysuccinimide ester) (DSP):6-mercapto-1-hexanol (MCH) mixed self-assembled monolayers (mSAMs) were formed on gold surfaces for immobilization of anti-thrombin antibody. The performance of the immunosensor was determined against the target protein thrombin at various concentrations using flow cell coupled SPR. The linear detection range of the immunosensor was 30.0–100.0 nM with an R2 value of 0,992. Limit of Detection (LOD) and Limit of Quantification (LOQ) were determined to be 6.0 nM and 30.0 nM, respectively. The selectivity of the immunosensor was tested against a non-target model protein, human serum albumin (HSA) and the obtained ΔRU value was found to be below the ΔRU value corresponding to the LOQ concentration for thrombin. The immunosensor’s capability to detect thrombin in diluted complex serum matrix was also tested and the obtained ΔRU value (159 ± 16) was compared with ΔRU value obtained for thrombin detection in PBS solution (137 ± 19). Based on the results, it was shown that DSP:MCH interface is a promising immobilization platform for binding biological recognition elements for the development of biosensors.

Determination of Eudragit® L100 in an Enteric-Coated Tablet Formulation Using Size-Exclusion Chromatography with Charged-Aerosol Detection.

Eudragit® L100 is a commonly used polymer in a coating layer of modified-release drug formulation to prevent drug release in the stomach. The amount of Eudragit® L100 in the formula determines the dissolution profile of drug at its release medium. Hence, its quantification in reference product will facilitate the formulation of a bioequivalent drug product. Some analytical methods including size-exclusion chromatography (SEC) have been reported for characterization of Eudragit® L100 either as single component or its conjugate with the enzyme, but none for its quantification in drug formulation. In this work, an SEC method with charged-aerosol detection (CAD) was developed for determination of Eudragit® L100 in an enteric-coated tablet formulation using Waters Ultrahydrogel 1000 and Waters Ultrahydrogel 120 columns in series. The mobile phase was a mixture of 90:10 (v/v) 44.75 mM aqueous ammonium acetate buffer, pH 6.6 and acetonitrile pumped at a constant flow rate of 0.8 mL/min in isocratic mode. The method was validated for specificity, working range, limit of detection (LOD), limit of quantification (LOQ), accuracy and precision. The method was shown to be specific for Eudragit® L100 against the diluent (mobile phase) and placebo of a coating layer for the tablet. A good correlation coefficient (r = 0.9997) of CAD response against Eudragit® L100 concentration from 0.1⁻1.0 mg/mL was obtained using polynomial regression. LOD and LOQ concentrations were 0.0015 and 0.0040 mg/mL, respectively. The mean recovery of Eudragit® L100 was in the range of 88.0⁻91.1% at three levels of working concentration: 50%, 100% and 150%. Six replicated preparations of samples showed good precision of the peak area with % relative standard deviation (RSD) 2.7. In conclusion, the method was suitable for quantification of Eudragit® L100 in an enteric-coated tablet formulation.

ANALYTICAL METHOD DEVELOPMENT AND VALIDATION FOR THE ESTIMATION OF IMATINIB MESYLATE AND ITS DIMER IMPURITY IN PHARMACEUTICAL FORMULATION BY REVERSE-PHASE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

Objective: The present study is to develop a simple, specific, and validated reverse-phase high-performance liquid chromatography (HPLC) method for the determination of imatinib mesylate and its dimer impurity in pharmaceutical dosage form.Methods: A HPLC instrument incorporated with column HiQ Sil C18 (250 mm × 4.6 mm, 5 μm), mobile phase as methanol and acetate buffer pH 3.5 in the ratio of 80:20 v/v was used for the determination of the imatinib mesylate and its dimer impurity. The detection wavelength was set at 273 nm. The flow rate of the mobile phase was 1.0 mL/min.Results: The retention time for imatinib mesylate was 8.060, and for dimer impurity, it was 11.398. The calibration plot was linear (R2=0.9971) and the % mean recoveries for imatinib mesylate were in the range of 99.83–101.57, and for dimer impurity, it was in the range of 98.16–99.18. The limit of detection concentration was found to be 0.570 μg/ml for imatinib mesylate and 0.033 μg/ml dimer impurity and limit of quantification concentration was 1.728 μg/ml for imatinib mesylate and 0.099 μg/ml dimer impurity.Conclusion: The projected method was validated and successfully functional for the estimation of imatinib mesylate and dimer impurity in formulations. It can be adopted apparently for routine quality control and research tests.

SIMULTANEOUS ANALYTICAL METHOD DEVELOPMENT OF 6-MERCAPTOPURINE AND 6-METHYLMERCAPTOPURINE IN DRIED BLOOD SPOT USING ULTRA PERFORMANCE LIQUID CHROMATOGRAPHY TANDEM MASS SPECTROMETRY

Objective: 6-mercaptopurine (6-MP) is a chemotherapeutic agent in the antimetabolite class. It has to go through the metabolic pathway to form6-methyl MP (6-MMP). This study aimed to obtain an optimum and validated method for the analysis of 6-MP and 6-MMP in dried blood spot (DBS)samples simultaneously and to evaluate the potential for future drug concentration monitoring in DBS samples.Methods: The quality control and calibration curves were made by spotting 40 μL blood on DBS paper and dried for 3 hrs. DBS papers were cut with adiameter of 8 mm and extracted with acetonitrile-methanol (1:3) containing internal standard 5-fluorouracil (5-FU). Separation was performed withwaters acquity ultra performance liquid chromatography BEH C18 column of 1.7 μm (2.1×100 mm) with a mobile phase consisting of 0.1% formicacid in water 0.1% formic acid in acetonitrile with gradient elution and a flow rate of 0.2 mL/minute. Mass detection was performed using WatersXevo TQD with positive electrospray ionization (ESI) for 6-MP and 6-MMP and negative ESI for 5-FU in the multiple reaction monitoring mode.Results: The detection rates of 6-MP, 6-MMP, and 5-FU were 153.09&gt;119.09, 167.17&gt;126.03, and 129.09&gt;42.05, respectively. This method was linearwith the range at 26-1000 ng/mL for 6-MP and 13-500 ng/mL for 6-MMP with consecutive r≥0.998 and ≥0.999, respectively. The % relative errorvalue and % relative standard deviation for accuracy and precision of intraday and interday were not more than 15% and not more than 20% at thelower limit of quantification concentration, respectively.Conclusions: This method fulfilled the requirements of selectivity, linearity, carry over, and matrix effects referring to the European Medicines Agencyguidelines.

A microbiological assay to estimate the antimicrobial activity of parenteral tildipirosin against foodborne pathogens and commensals in the colon of beef cattle and pigs.

Tildipirosin (TIP) is a novel 16-membered-ring macrolide authorized for the treatment of bovine and swine respiratory disease. The pH dependency of macrolide antimicrobial activity is well known. Considering that the pH in the colon contents of growing beef cattle and pigs is usually below pH 7.0, the minimum inhibitory concentrations (MIC) of TIP against foodborne bacterial pathogens such as Campylobacter (C.) coli, C. jejuni and Salmonella enterica and commensal species including Enterococcus (E.) faecalis, E. faecium and Escherichia coli were determined under standard (pH 7.3 ± 1) or neutral as well as slightly acidic conditions. A decrease in pH from 7.3 to 6.7 resulted in an increase in MICs of TIP. Except for the MICs > 256 μg/mL observed in the resistant subpopulation of the C. coli and the Enterococcus species, the MIC ranges increased from 2-8 μg/mL to 64-> 256 μg/mL for Salmonella enterica and E. coli, from 8-16 μg/mL to 32-128 μg/mL for the two Campylobacter species, and from 4-32 μg/mL to 128-> 256 μg/mL for both Enterococcus species. To estimate the antimicrobial activity of TIP in the colon contents of livestock during recommended usage of the parenterally administered TIP (Zuprevo(®) ), and to compare this with the increased MICs at the slightly acidic colonic pH, we developed and validated a microbiological assay for TIP and used this to test incurred faecal samples collected from cattle and pigs. Microbiological activity of luminal TIP was determined in aqueous supernatants from diluted faeces, using standard curves produced from TIP-spiked faecal supernatants. The limit of quantification (LOQ) for TIP was 1 μg/mL (ppm). In a cattle study (n = 14), 3 of 28 faecal samples collected 24 and 48 h post-treatment were found to contain TIP above the LOQ (concentrations of 1.3-1.8 ppm). In another cattle study (n = 12) with faecal samples collected at 8, 24 and 48 h post-treatment, TIP concentrations were above the LOQ in 4 of the 8 h samples (1.2-2.6 ppm) and one of the 24-h samples (1.3 ppm). In a pig study (n = 12) with faecal samples collected 24, 48 and 72 h post-treatment, only one sample contained TIP above the LOQ (concentration 1.5 ppm). In another pig study (n = 12), with samples collected at 8, 24 48 and 96 h post-treatment, TIP concentrations were above the LOQ in one 8-h sample (1.1 ppm) and two 24-h samples (2.3 and 2.5 ppm). None of the 48-h and 96-h samples from these 4 studies contained measurable TIP concentrations. Thus, in cattle and pigs, only a small fraction of faecal samples collected up to 24 h postdosing contained measurable microbiologically active TIP, with its maximum limited to 2.6 μg/mL. This is several log2 dilution steps below the MICs of TIP against foodborne pathogens and commensals collected under acidic conditions comparable with those in the colonic contents and may explain a lack of intestinal dysbacteriosis with parenteral tildipirosin in livestock.

Evaluation of Pharmaceutical Quality of Mesalamine Delayed Release Tablets Using a New High Sensitivity Reversed-Phase UPLC Method for its Genotoxic/Aniline Impurity

A reversed phase ultra performance liquid chromatography (UPLC) method was developed and validated for the quantification of aniline in mesalamine delayed-release tablets. The optimization of the experimental condition was carried out considering some important requirements like, detection limit, short run time and reproducibility. In the present study, isocratic reversed-phase UPLC method was developed for determination and separation of aniline from the drug product. The drug and impurity are well separated by using a reversed phase (Reprosil Gold C18-XBD) column and mobile phase comprising of buffer pH 6.0 and acetonitrile in the ratio of 90:10 v/v. Other UPLC parameters which were optimised are flow rate, 0.5 mL/min; detection wavelength, 200 nm; column oven temperature, 50°C and injection volume 7 µL. Stability indicating capability was also established by forced degradation experiments. The method was validated as per ICH guideline. LOQ (limit of quantification) concentration (18 ng/mL) was found precise with RSD of less than 2%. In essence, the present study provides an improved low detection limit and lower run time for evaluation of pharmaceutical quality of mesalamine delayed-release formulation. Moreover, the developed method was also successfully applied for quantification of aniline in mesalamine delayed-release formulation. The same method can also be used for determination of aniline from drug substances.

Occurrence of Pharmaceuticals in River Water and Their Elimination in a Pilot-Scale Drinking Water Treatment Plant

The occurrence of four beta blockers, one antiepileptic drug, one lipid regulator, four anti-inflammatories, and three fluoroquinolones was studied in a river receiving sewage effluents. All compounds but two of the fluoroquinolones were observed in the water above their limit of quantification concentrations. The highest concentrations (up to 107 ng L(-1)) of the compounds were measured during the winter months. The river water was passed to a pilot-scale drinking water treatment plant, and the elimination of the pharmaceuticals was followed during the treatment. The processes applied by the plant consisted of ferric salt coagulation, rapid sand filtration, ozonation, two-stage granular activated carbon filtration (GAC), and UV disinfection. Following the coagulation, sedimentation, and rapid sand filtration, the studied pharmaceuticals were found to be eliminated only by an average of 13%. An efficient elimination was found to take place during ozonation at an ozone dose of about 1 mg L(-1) (i.e., 0.2-0.4 mg of O3/ mg of TOC). Following this treatment, the concentrations of the pharmaceuticals dropped to below the quantification limits with the exception of ciprofloxacin. Atenolol, sotalol, and ciprofloxacin, the most hydrophilic of the studied pharmaceuticals, were not fully eliminated during the GAC filtrations. All in all, the treatment train was found to very effectively eliminate the pharmaceuticals from the rawwater. The only compound that was found to pass almost unaffected through all the treatment steps was ciprofloxacin.

Validated Chiral LC Method for the Enantiomeric Separation of β-Amino-β-(3-Methoxyphenyl) Propionic Acid

A chiral liquid chromatographic method for enantiomeric resolution of β-amino-β-(3-methoxyphenyl) propionic acid was developed and validated. The “hybrid” π-electron donor–acceptor based stationary phase (R,R) Whelk-01 was found to be enantiomerically selective for (R) and (S) enantiomers of β-amino-β-(3-methoxyphenyl) propionic acid with a resolution greater than 2.0. The effects of isopropyl alcohol and ethanol on enantioselectivity and resolution of enantiomers were evaluated. Calibration curves were linear over the range of 0.10–1.00, with a regression coefficient (r) of 0.999. The limit of detection (LOD) and limit of quantification (LOQ) were 300 and 1,000 ng mL−1 respectively for 10 μL injection volume. The percentage RSD of the peak area of six replicate injections of (S) enantiomer at LOQ concentration was 2.8. The percentage recovery of (S) enantiomer from (R) enantiomer samples ranged from 92 to 102. The test solution was observed to be stable up to 24 h after the preparation. The developed normal phase chiral LC method can be used for the enantiomeric purity evaluation of R-β-amino-β-(3-methoxyphenyl) propionic acid.

Validated Reversed Phase HPLC Method for the Analysis of the Food Additive, Sodium Benzoate, in Soft Drinks and Jams

A rapid, simple, and sensitive method is described for the determination of the food preservative sodium benzoate in soft drinks, various jams, and ketchup. The method utilizes high performance liquid chromatography followed by diode array detection. Chromatographic separation was achieved using a C18 reversed phase column and methanol: water (70∶30, v/v) adjusted to pH 3.45 with glacial acetic acid as mobile phase, 0.45 mL . min−1 flow rate, and UV detection at 245 nm. Amoxicillin was used as the internal standard. The retention time observed for sodium benzoate and amoxicillin were 5.01 and 12.07 min, respectively. The method is selective, reliable, and reproducible with a relative standard deviation of 0.66 and linear in the range of 50–450 ng/mL sodium benzoate concentration. The limit of detection and limit of quantification concentrations were 61 pg/mL and 203 pg/mL, respectively. The proposed method can be used for the routine analysis of sodium benzoate in soft drinks and jams.

Simplified ultraviolet liquid chromatographic method for determination of sertindole, dehydrosertindole and norsertindole, in human plasma

An ultra-violet high-performance liquid chromatographic method was developed for the determination of sertindole, an atypical antipsychotic drug and its main metabolites dehydrosertindole and norsertindole, in human plasma. With a small sample volume, after a single-step liquid–liquid extraction, the compounds were separated on a reversed-phase XTerra ® RP 18 column, eluted with 45% of acetonitrile and 55% of ammonium acetate buffer (0.05 M, adjusted pH 8) and detected at 256 nm within 11 min. This method shows a good linearity for plasma concentration between 5–100 ng/ml and 100–1000 ng/ml, a good precision (inter and intra day CV < 11%) and a good inter-assay accuracy (bias < 11%). The limit of quantification concentration was 5 ng/ml. The absolute recovery of sertindole was higher than 99%. This rapid and sensitive method could be used for therapeutic drug monitoring as well as for overdose management.