bluuebunny/crossref_metadata_2025 huggingface dataset:
Checking the Validity of the Harvesting and Disaggregating Steps in Laboratory Tests of Surface Disinfectants
Authors: Hamilton Martin A , Buckingham-Meyer Kelli , Goeres Darla M
Published: February 2020

A chemical disinfectant against surface-associated bacteria typically uses carriers (e.g., glass disks) that are purposely contaminated with bacteria prior to disinfection. After disinfection, the bacteria are harvested by mechanically separating them from the carrier surface to form a suspension of cells in a dilution tube. Bacterial clumps in the tube are disaggregated using mechanical or chemical techniques, thereby creating a well-mixed suspension of single cells suitable for enumeration. Efficacy is quantified by comparing the viable cell count for a disinfected carrier to the viable cell count for sham-disinfected (control) carrier. A test is said to be biased (invalid) if the observed efficacy measure is systematically higher or lower than the true efficacy. It is shown here for the first time that the bias attributable to the harvesting and disaggregating steps of a disinfectant test can be measured. For some conventional biofilm harvesting and disaggregating techniques, laboratory checks showed either negligible bias or important bias, depending on the disinfectant. Quantitative bias checks on the harvesting and disaggregating steps are prudent for each combination of carrier material, microorganism, and disinfectant. The quantitative results should be augmented by microscopic examination of harvested disinfected and control carriers and of the disaggregated suspensions.
Characterization of Hapten-Protein Conjugates: Antibody Generation and Immunoassay Development for Chlorophenoxyacetic Acid Pesticides
Authors: Boro Robin C , Singh K Vikas , Suri C Raman
Published: February 2020

The generation of specific and sensitive antibodies against small molecules is greatly dependent upon the characteristics of the hapten-protein conjugates. In this study, we report a new fluorescence-based method for the characterization of hapten-protein conjugates. The method is based on an effect promoted by hapten-protein conjugation density upon the fluorescence intensity of the intrinsic tryptophan chromophore molecules of the protein. The proposed methodology is applied to quantify the hapten-protein conjugation density for two different chlorophenoxyacetic acid pesticides, 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4-dichlorophenoxybutyric acid (2,4-DB), coupled to carrier protein. Highly sensitive anti-2,4-D and anti-2,4-DB antibodies were obtained using these well-characterized hapten-protein conjugates. The generated antibodies were used in an immunoassay format demonstrating inhibitory concentration (IC50) values equal to 30 and 7 ng/mL for 2,4-D and 2,4-DB, respectively. Linearity was observed in the concentration range between 0.1500 ng/mL with LODs around 4 and 3 ng/mL for 2,4-D and 2,4-DB, respectively, in standard water samples. The proposed method was successfully applied for the determination of the extent of hapten-protein conjugation to produce specific antibodies for immunoassay development against pesticides.
Levels of Bisphenol A Diglycidyl Ether (BADGE) and Bisphenol F Diglycidyl Ether (BFDGE) in Canned Liquid Infant Formula Products in Canada and Dietary Intake Estimates
Authors: Cao Xu-Liang , Dufresne Guy , Clement Genevieve , Blisle Stphane , Robichaud Andr , Beraldin Franca
Published: February 2020

A method based on solid-phase extraction followed by HPLC analysis with fluorescence detection was developed for the determination of bisphenol A diglycidyl ether (BADGE) and bisphenol F diglycidyl ether (BFDGE) in liquid infant formula. In this method, instead of trying to isolate and measure each individual form of the molecules, hydrolysis of BADGE, BFDGE, BADGEH2O, and BFDGEH2O was forced to completion to their stable forms, BADGE2H2O and BFDGE2H2O, before extraction. The method LODs were 2.0 ng/g for BADGE and 3.0 ng/g for BFDGE. Extraction recoveries were 6191 for BADGE, and 5582 for BFDGE over the concentration range of 10 to 50 ng/g. The method was used to analyze samples of 21 canned liquid infant formula products for BADGE and BFDGE. BADGE was detected in samples of all products at levels ranging from as low as 2.4 ng/g to as high as 262 ng/g. BFDGE was detected in only one product (40 ng/g), and this product also had the highest BADGE level (262 ng/g). HPLC/MS/MS with a similar LOD was also used to confirm the results. The probable daily intakes (PDI) of BADGE and BFDGE due to consumption of canned liquid infant formula were estimated for infants from premature to 1218 months of age. The maximum PDI of BADGE was 22 g/kg body weight/day for the 1218 months old with the maximum formula intake. The maximum PDI of BFDGE was <3.4 g/kg body weight/day.
Simultaneous Spectrophotometric Determination of Benzoic Acid, Sorbic Acid, and Ascorbic Acid Using a Net Analyte Signal-Based Method
Authors: Naseri Abdolhossein , Ghorbani-Kalhor Ebrahim , Vallipour Javad , Jafari Samira , Shahverdizadeh Gholam Hossein , Asadpour-Zeynali Karim
Published: February 2020

The net analyte preprocessing/classical least-squares (NAP/CLS) method is a simple chemometric method that has been used for the simultaneous spectrophotometric determination of benzoic acid, sorbic acid, and ascorbic acid. The obtained results indicated that the performances of the NAP/CLS and partial least-squares methods were almost identical. The net analyte signal (NAS) concept was also used to calculate multivariate analytical figures of merit, such as LOD, selectivity, and sensitivity. Wavelength selection was applied based on the concept of NAS regression, and improved the method performance in samples containing nonmodeled interferences. The method afforded recoveries in the range of 98105. The proposed method was successfully applied to determination of the analytes in an Iranian soft drink.
GeneQuence Salmonella Assay
Authors: Alles Susan , Mozola Mark , Hammack Thomas
Published: February 2020

A study was conducted to validate the GeneQuence Salmonella DNA hybridization assay, Performance Tested Method 030201, for detection of Salmonella spp. in peanut butter. The study was organized by the AOAC Research Institute under its Emergency Response Validation program. Peanut butter samples inoculated with S. Typhimurium were prepared by an independent laboratory and shipped to study participants for testing. The set of blind-coded test samples consisted of five uninoculated controls, 20 portions inoculated with S. Typhimurium at a low level [determined by most probable number (MPN) analysis to contain 1.1 CFU/25 g portion], and 20 portions inoculated with S. Typhimurium at a higher level (11 CFU/25 g portion by MPN analysis). Samples were tested in parallel by the GeneQuence method and by the U.S. Food and Drug Administration's Bacteriological Analytical Manual reference culture procedure. All five control samples were negative by both methods. For the low-level samples, the same two samples were positive by both the GeneQuence and reference methods. For the high-level samples, the same 19 samples were positive by both methods. All positive GeneQuence assays were confirmed by plating from associated broth cultures. Agreement between the GeneQuence and reference methods was 100. Sensitivity and specificity of the GeneQuence method were both 100. Because neither the low- nor the high-level samples yielded the desired fractional positive results (515 positives out of 20 samples), a second trial was conducted. Samples in the second trial contained 0.1 and 0.5 CFU/25 g portion for the low and high levels, respectively. All five control samples were negative by both methods. For the low-level samples, the same two samples were positive by both the GeneQuence and reference methods. For the high-level samples, the same three samples were positive by both methods. All positive GeneQuence assays were confirmed by plating from associated broth cultures. Sensitivity and specificity of the GeneQuence method were both 100. Although once again the desired level of fractional positive results was not obtained, there was 100 agreement between the GeneQuence and reference methods. Based on the results of both trials, it is recommended that the validated claims for Performance Tested Method 030201 be expanded to include peanut butter.
iQ-Check Salmonella II: Real-Time Polymerase Chain Reaction Test Kit
Authors: Lauer Wendy F , Sidi Caroline D , Tourniaire Jean-Philippe , Hammack Thomas
Published: February 2020

iQ-Check Salmonella II is a real-time PCR kit for detection of Salmonella in foods. Specific oligonucleotide probes are used to detect target DNA during the amplification, by hybridizing to the amplicons. These probes are linked to a fluorophore, which fluoresces only when hybridized to the target sequence. As part of an Emergency Response Validation due to a massive outbreak and subsequent recall, peanut butter was tested to compare the performance of iQ-Check Salmonella II to the U.S. Food and Drug Administration's Bacteriological Analytical Manual (FDA-BAM) reference method for detection of Salmonella. A single enrichment in buffered peptone water was used for a reduced enrichment time of 21 1 h over the 48 h reference method. There was no significant difference in the performance of the iQ-Check kit when compared to the FDA-BAM method, as determined by Chi-square analysis. All samples identified as positive by iQ-Check were confirmed by reference method protocol.
RAPID' Salmonella Chromogenic Medium
Authors: Lauer Wendy F , Martinez Frederic L , Hammack Thomas
Published: February 2020

RAPID' Salmonella is a chromogenic medium for isolation and detection of Salmonella spp. in food, based on two enzymatic activities. All presumptive Salmonella-positive colonies are magenta, including lactose-positive Salmonella. S. Typhi, and S. Paratyphi serotypes, due to detection of C8 esterase activity. In order to differentiate Salmonella from other Enterobacteriaceae, the medium includes a second chromogenic substrate. As part of an Emergency Response Validation due to a massive outbreak and subsequent recall, peanut butter was tested to compare the performance of RAPID' Salmonella to the U.S. Food and Drug Administration's Bacteriological Analytical Manual reference method for detection of Salmonella. Sensitivity and specificity for RAPID' Salmonella were 100.
Quantification of Paroxetine in Human Plasma by Liquid Chromatography Coupled with Electrospray Ionization Tandem Mass Spectrometry
Authors: Shah Hiten J , Kundlik Mohan L , Kakad Abhijit , Patel Nitesh K , Pandya Ankit , Khatri Vanita , Prajapati Shivkumat , Subbaiah Gunta , Patel Chhagan N
Published: February 2020

A rapid LC coupled with electrospray ionization (ESI) MS/MS method was developed and validated for the quantification of paroxetine in heparinized human plasma. The plasma samples were prepared by the solid-phase extraction method without drying or reconstitution. Elution was done with 0.5 mL 0.2 (v/v) formic acid in methanolacetonitrile (65 + 35, v/v). The analyte and the internal standard (IS; imipramine hydrochloride) were chromatographed on a BDS Hypersil C18 column. The analyte was analyzed by LC/MS/MS with only 1.7 min run time. An ESI interface was chosen for ionization of the analyte from the sample matrix. Selected reaction monitoring mode for detection of paroxetine and the IS were achieved by using m/z 330.17/192.10 and 281.13/86.14, respectively. The LC retention times for paroxetine and imipramine were 0.94 and 1.05 min, respectively. The method was linear in the concentration range of 0.580.0 ng/mL with r ≥0.9995. Recovery of paroxetine and imipramine ranged from 90 to 95. The assay has been successfully applied to bioequivalence study samples for estimation of paroxetine in healthy human subjects.
Evaluation of the Assurance GDS® for Salmonella Method in Foods and Environmental Surfaces: Multilaboratory Collaborative Study
Authors: Feldsine Philip T , Jucker Markus T , Kaur Mandeep , Lienau Andrew H , Kerr David E ,   Collaborators:, Adamson A , Beaupre L , Bishop J , Casasola E , Cote C , Desilets S , D’lima C , Elahimanesh P , Fitzgerald S , Forgey R , Fortin J , Gohil V , Griffin J , Hardin M , Kaur D , Ketrenos J , King A , Kupski B , Luce S , Lucia L , Maeda S , Markun D , Marquez-Gonzalez M , McClendon J , McKessock J , Nelson C , Nguyen T , O’Brien C , Ramos M , Reilly S , Roa N , Schiffelbein Z , Shaffer K , Shepherd D , Sowell S , Trujillo E , Wang S , Williams K
Published: February 2020

A multilaboratory collaborative study was conducted to compare the detection of Salmonella by the Assurance GDS® for Salmonella method and the Reference culture methods. Six foods, representing a variety of low microbial and high microbial load foods were analyzed. Seventeen laboratories in the United States and Canada participated in this study. No statistical differences (P < 0.05) were observed between the Assurance GDS for Salmonella and the Reference culture methods for any inoculation level of any food type or naturally contaminated food, except for pasta, for which the Assurance GDS method had a higher number of confirmed test portions for Salmonella compared to the Reference method.
Determination of Ethoxyquin by High-Performance Liquid Chromatography with Fluorescence Detection and Its Application to the Survey of Residues in Food Products of Animal Origin
Authors: Aoki Yoichi , Kotani Akira , Miyazawa Naomi , Uchida Kazunari , Igarashi Yu , Hirayama Norio , Hakamata Hideki , Kusu Fumiyo
Published: February 2020

An analytical method using HPLC with fluorescence detection (HPLC-FL) has been developed and applied for the survey of residue levels of ethoxyquin in a variety of food products of animal origin. HPLC was performed using a silica octadecylsilane column, butylhydroxytolueneacetonitrilewater (0.05 + 800 + 200, v/v/v) mobile phase, and detection at excitation and emission wavelengths of 370 and 415 nm, respectively. HPLC/MS was used to confirm whether a chromatographic peak was ethoxyquin. The LOQ of the foods was 0.01 µg/g, except for pig fat and cow's milk, and the RSD (n = 6) at 0.1 µg/mL of the standard solution was 1.12. The accuracy of the calculated data of the standard solution was within the range of 94.0 to 101.2. Recoveries of ethoxyquin from the food products of cattle, pigs, chickens, and salmon were more than 71.0 with an RSD of <9.3, except for chicken liver at different concentration levels, including the lower LOQ, the maximum residue limit (MRL), and in some tissues, twice the MRL. Residue levels of ethoxyquin in 33 commercially available food products of animal origin that were purchased on the west side of the Tokyo metropolitan area were surveyed. Contents of ethoxyquin residues in three chicken fat samples by the HPLC-FL method were 0.08, 0.03, and 0.04 µg/g, all less than the MRL (5 µg/g).
Trace-Level Detection of Atrazine Using Immuno-Chemiluminescence: Dipstick and Automated Flow Injection Analyses Formats
Authors: Chouhan Raghuraj S , Rana Kumar Vikash S , Suri C Raman , Thampi Ravindranathan K , Thakur Munna S
Published: February 2020

A sensitive chemiluminescence (CL)-based immunoassay technique based on both dipstick and flow injection analytical formats is reported for the detection of atrazine. In the dipstick-based immunoassay technique, antibody (anti-atrazine) was first immobilized on the nitrocellulose membranes. The dipstick was then treated with atrazine and atrazine-horseradish peroxidase conjugate (atra-HRP) to facilitate the competitive binding. The dipstick was further treated with urea-hydrogen peroxide (U-H2O2) and luminol to generate photons. The number of photons generated was inversely proportional to the atrazine concentration. In the flow injection analysis (FIA) format, the antibody was immobilized on protein-A sepharose matrix and packed in a glass capillary column, which functioned as an immunoreactor. Competitive binding of antigen and antibody occurred. The CL signals generated during the biochemical reactions were correlated with atrazine concentrations in the analytical samples. By using dipstick technique, it was possible to detect atrazine concentration down to 0.1 ng/mL; with the FIA format, the detection of atrazine was down to 0.01 ng/mL.
Immunoassays for the Rapid Detection of Gentamicin and Micronomicin in Swine Muscle
Authors: Chen Yiqiang , Li Xiangmei , He Lidong , Tang Shuheng , Xiao Xilong
Published: February 2020

A monoclonal antibody (mAb)-based ELISA and strip test for gentamicin (GEN) and its analogue micronomicin (MIN), are reported in this study. The conjugate gentamicin-glutaraldehyde-bovine serum albumin (GEN-GDA-BSA) was used as an immunogen. The produced anti-GEN mAB exhibited high cross-reactivity with micronomicin (MIN; 131.2) and slight or negligible cross-reactivity with other aminoglycosides. Based on this mAB, an ELISA and a strip test for GEN and MIN were developed and evaluated. The ELISA showed a 50 inhibition concentration (IC50) of 0.75 ng/mL for GEN and 0.58 ng/mL for MIN. For GEN, the average recoveries at 25200 µg/kg ranged from 73 to 91, with intraday CVs of 916 and interday CVs of 815. For MIN, the average recoveries ranged from 108 to 131, with intraday CVs of 1016 and interday CVs of 815. In contrast, the strip test for GEN or MIN had a detection limit of 5 ng/mL in phosphate-buffered saline and 50 µg/kg in muscle (n = 24), and the results could be judged within 10 min. The detection results of incurred samples analyzed by the strip test, ELISA, and HPLC indicated that the two immunoassays correlated well with the HPLC method and could be used as convenient tools for the rapid screening of GEN and MIN residues in swine muscle.
Confirmation of Hormones in Animal Serum by Liquid Chromatography/Tandem Mass Spectrometry According to European Commission Decision 2002/657
Authors: McDonald Mark , Malone Edward , McBride John
Published: February 2020

A novel and rapid method was developed and validated for the confirmation of endogenous and synthetic hormones in animal serum using LC/MS/MS. Detection of 17 β-estradiol and β-testosterone below the respective European Union-recommended levels of 0.1 and 0.5 µg/L was achieved, as was a required performance level of 0.1 µg/L for 17 α-estradiol and 0.5 µg/L for 17 α-testosterone, medroxyprogesterone-17-acetate, and progesterone. The method was established with dilution of serum followed by ion-exchange SPE, LC separation and MS detection with electrospray ionization, selected reaction monitoring, and positive/negative switching. Two characteristic transitions were monitored for each analyte. The method was applied to bovine, ovine, porcine, equine, and avian samples and validated according to European Commission Decision 2002/657/EC and accepted for ISO/IEC 17025:2005 accreditation. An extended calibration curve allows naturally occurring levels of endogenous hormones to be quantified. Recoveries ranged from 97.3 for 17 α-testosterone to 102.0 for 17 α-estradiol. The decision limit CCα ranged from 0.02 µg/L for 17 α- and β-estradiol to 0.12 µg/L for progesterone. Detection capability CCβ ranged from 0.03 µg/L for 17 -estradiol to 0.20 µg/L for progesterone.
A System for Repertoire Cloning and Phage Display of Murine and Leporid Antibody Fragments
Authors: Schüller Carolin , Wiebe Julia C , Pegel Antje , Kramer Karl , Skerra Arne , Hock Bertold
Published: February 2020

Even though rabbit antibodies (Abs) are known to exceed murine Abs with respect to specificity, affinity, and stability, cloned leporid immune repertoires have been rarely considered in recombinant Ab preparation for environmental analysis. We have developed a set of four tetp/o-based phasmid vectors that allow the efficient cloning of both murine and leporid Ab repertoires. These vectors differ in the design of the cloning sites, choice of signal peptides, and antibiotic selection markers. A set of 39 primer oligodeoxynucleotides has been developed for the PCR amplification of rabbit Ab genes, representing the most exhaustive coverage of the leporid immune repertoire described so far. The atrazine-specific murine Fab fragment K411B and a cloned V-gene repertoire from sulfonamide-immunized rabbits were used to compare these phasmids with respect to expression of Fab fragments, phagemid titers, and number of Fab displaying phagemid particles. Our results show that the ratio of recombinant phagemids could be increased up to 65% of total phage titer by utilizing the appropriate phasmid. Based on this system, the selection of two sulfonamide-specific rabbit Abs, SA2 23 and SA2 90, was accomplished after a single phagemid panning round.
Determination of Total Procyanidins in Selected Chocolate and Confectionery Products Using DMAC
Authors: Payne Mark J , Hurst William Jeffrey , Stuart David A , Ou Boxin , Fan Ellen , Ji Hongping , Kou Yan
Published: February 2020

A simple, specific, high-throughput colorimetric method based on the reaction of 4-dimethylaminocinnamaldehyde (DMAC) with flavan-3-ols was developed to determine total procyanidins in selected cacao-based products. Extracts of defatted samples were dispensed into a 96-well plate and reacted with DMAC. The absorbance of the reaction products was measured at 640 nm and compared to commercially available procyanidin B2 as a standard. The use of the 96-well plates and a plate reader dramatically improved sample throughput. A standard protocol was established and used for further studies. The calibration was found to be linear from 1100 ppm. The DMAC reagent reacted relatively specifically to (–)-epicatechin, (+)-catechin, epigallocatechin, gallocatechin, the gallates of catechin, epicatechin, gallocatechin, and epigallocatechin, oligomeric procyanidins of cocoa up to n = 4, and A-type procyanidins. Little or no reaction occurred with cyanidins and representative compounds of phenolic acids, flavones, flavanones, flavonols, anthocyanidins, isoflavones, and stilbenes. Sample precision studies were carried out on 10 different test materials over several weeks, and yielded RSD values of 4.0 to 9.5%. The method was ring-tested in three laboratories using blinded test materials including cocoa beans, cocoa powder, chocolate liquor, dark chocolate, and milk chocolate. There was excellent agreement of the results between laboratories.
Efficiency Evaluation of the Main Multiresidue Methods Used in Europe for the Analysis of Amitraz and Its Major Metabolites
Authors: Ferrer Carmen , Agüera Ana , Mezcua Milagros , Fernández-Alba Amadeo R , Mack Dorothea , Anastassiades Michelangelo , Gamón Miguel
Published: February 2020

This paper compares the performance of the three most widely employed multiresidue methods [quick, easy, cheap, effective, rugged, and safe (QuEChERS), mini-Luke, and ethyl acetate] currently used for the determination of amitraz residues in fruits. A fast and differentiated analysis of amitraz and its two main metabolites, N-2,4-dimethylphenyl-N-methylformamidine and 2,4-dimethylformanilide, was performed by HPLC-electrospray ionization-MS/MS using a triple quadrupole mass spectrometer in the positive mode. A test of the stability of the standard solutions showed a rapid hydrolysis of amitraz to the amide and amidine derivatives in solutions containing water, including QuEChERS extracts of crops that were previously acidified. Two useful mass transitions were used to confirm the presence of each analyte in the sample extracts. LOD values ranging from 0.4 to 2.0 g/kg were obtained. Linearity of response over 2 orders of magnitude was demonstrated (r2 > 0.999) in solvent and pear extract. The recovery studies were performed on pear blanks spiked at two concentration levels, 50 and 500 g/kg (N 5). Best recoveries, ranging from 75 to 103, were obtained by the application of the QuEChERS method with CV <8 in all cases. The QuEChERS method was applied to a monitoring study carried out by the Chemical and Veterinary Investigation Office Stuttgart laboratory. From the 63 pear samples analyzed, 21 contained amitraz residues (expressed as sum) ranging from 0.02 to 2.9 mg/kg. Amitraz parent was detected only in a few cases at very low concentration levels, with N-2,4-dimethylphenyl-N-methylformamidine being the metabolite almost entirely representing the total residue. These results emphasize that the residue situation is clearly underestimated if only the parent compound is targeted, and they reinforce how important it is to include amitraz in the target scope of pesticide residue laboratories, especially since the concentrations detected exceeded the Acute Reference Dose in the majority of cases and pose a health risk to the consumer.
Determination of Tadalafil in Pure Powder and Tablet Dosage Form by High-Performance Liquid Chromatography
Authors: Barot Tushar G , Patel Popatbhai K
Published: February 2020

A simple and accurate method to determine tadalafil (TAD) in pure powder and tablet dosage form was developed and validated using HPLC. The separation was achieved on an Xterra RP18 column (150 4.6 mm id, 3.5 m) in the isocratic mode using bufferacetonitrile (70 + 30, v/v), adjusted to pH 7.00 0.05 with triethylamine as the mobile phase at a flow rate of 1.0 mL/min. The photodiode array detector was set at 225 nm. Quantification was achieved over the concentration range of 50.7152.10 g/mL with mean recovery of 100.26 0.75. The method was validated and found to be simple, accurate, precise, and specific. The method was successfully applied for the determination of TAD in pure powder and tablet dosage form without interference from common excipients or degradation products.
Validation of a Stability-Indicating RP-HPLC Method for the Determination of Entecavir in Tablet Dosage Form
Authors: Dalmora Sérgio Luiz , Sangoi Maximiliano da Silva , Nogueira Daniele Rubert , Silva Lucélia Magalhães da
Published: February 2020

An RP-HPLC method was validated for the determination of entecavir in tablet dosage form. The HPLC method was carried out on a Gemini C18 column (150 4.6 mm id) maintained at 30C. The mobile phase consisted of acetonitrilewater (95 + 5, v/v)/potassium phosphate buffer (0.01 M, pH 4; 9 + 91, v/v) pumped at a flow rate of 1.0 mL/min. Photodiode array detection was at 253 nm. The chromatographic separation was obtained with a retention time of 4.18 min, and the method was linear in the range of 0.5200 g/mL (r2 0.9998). The specificity and stability-indicating capability of the method was proven through forced degradation studies, which also showed that there was no interference of the excipients and an increase of the cytotoxicity only by the basic condition. The accuracy was 101.19, with bias lower than 1.81. The LOD and LOQ were 0.39 and 0.5 g/mL, respectively. Method validation demonstrated acceptable results for precision and robustness. The proposed method was applied for the analysis of tablet formulations, to improve QC and assure therapeutic efficacy.
TEMPO® EC for the Enumeration of Escherichia coli in Foods: Collaborative Study
Authors: Crowley Erin , Bird Patrick , Torontali Marianne , Goetz Katherine , Agin James , Goins David , Johnson Ronald ,   Collaborators:, Achen M , Barlowe A , Clark M , Colón-Reveles J , Dixon K , Fisher K , Hanson P , Jechorek R , Johnson L , Kelly M , Kim S , Kohler H , Kondratko D , Kupski B , McCallum K , Mills J , Mohnke F , Moon B , Olson B , Reed C , Sauter J , Thompson L
Published: February 2020

The automated method for enumeration of Escherichia coli, TEMPO EC, in foods uses a dehydrated culture medium and enumeration card containing 48 wells across three different dilutions for the automatic determination of the most probable number (MPN). The alternative method was compared in a multilaboratory collaborative study to AOAC Official MethodSM 966.24. Six food types were artificially contaminated with E. coli: raw ground beef, bagged lettuce, cooked chicken, pasteurized crabmeat, frozen green beans, and pasteurized whole milk. All foods were analyzed for E. coli counts by 11 collaborating laboratories throughout the United States. Test portions from the six food types each contaminated at four different contamination levels were evaluated. The study demonstrated that the TEMPO EC method is a reliable, automated assay for the enumeration of E. coli in foods.
Finding and Confirming Nontargeted Pesticides Using GC/MS, LC/Quadrupole-Time-of-Flight MS, and Databases
Authors: Meng Chin-Kai , Zweigenbaum Jerry , Fürst Peter , Blanke Eva
Published: February 2020

Two methods are described for identifying unknowns in complex matrixes. One approach, suitable for nonpolar and thermally stable compounds, uses GC/MS with a deconvolution technique, followed by a spectral library search. The second approach, suitable for polar and thermally labile compounds, uses LC/quadrupole-time-of-flight (LC/Q-TOF) MS with a Molecular Feature Extractor (MFE), followed by a database search on the exact mass of each found compound. Two example analyses are presented to illustrate the GC/MS method. The first example processes data files obtained by a multiresidue analysis of pesticides in surface water. In this example, 71 pesticides were identified using Deconvolution Reporting Software in conjunction with Automated Mass Spectral Deconvolution and Identification System software. The second example illustrates the deconvolution approach in detail using peaches and pears analyzed after extraction by the quick, easy, cheap, effective, rugged, and safe (QuEChERS) protocol. The LC/Q-TOF approach is illustrated through analysis of a grape extract prepared by the QuEChERS protocol. The resulting total ion chromatogram was found by the MFE to contain 510 chemicals (components). The masses of found chemicals were subsequently searched against an Exact Mass Database of hundreds pesticides, and 15 exact mass hits were found. Several of these exact mass hits were selected for further confirmation by MS/MS analysis using the same LC/Q-TOF.
Determination of Hydrogen Cyanide in Cigarette Mainstream Smoke by LC/MS/MS
Authors: Mottier Nicolas , Jeanneret Florent , Rotach Michel
Published: February 2020

An LC/MS/MS method is presented for the determination of hydrogen cyanide in cigarette mainstream smoke. Cyanide is derivatized with 2,3-naphthalenedicarboxaldehyde and taurine to form a benzo[f]isoindole derivative, which is then analyzed by LC/MS/MS. Isotopic KCN (K13C15N) was used as an internal standard. The regression equation was linear within the range 2.4331 ng/mL for cyanide with a correlation coefficient >0.999. The LOD was calculated as 4.1 ng/cigarette. The influence of the sodium hydroxide trapping solution concentration on the results is discussed. A 1 M solution showed the best results in terms of sample stability and trapping efficiency. The method proved to be robust, reliable, and more selective than current methods, making it a logical choice for determination of total cyanide in cigarette smoke.
Validated HPTLC Method for Simultaneous Quantitation of Paracetamol, Diclofenac Potassium, and Famotidine in Tablet Formulation
Authors: Khatal Laxman D , Kamble Asmita Y , Mahadik Mahadeo V , Dhaneshwar Sunil R
Published: February 2020

A sensitive, simple, selective, precise, and accurate HPTLC method of analysis for paracetamol, diclofenac potassium, and famotidine both as a bulk drug and in tablet formulation was developed and validated. The method used HPTLC aluminum plates precoated with silica gel 60F254 as the stationary phase, and the mobile phase consisted of tolueneacetonemethanolformic acid (5 + 2 + 2 + 0.01, v/v/v/v). Densitometric evaluation of the separated zones was performed at 274 nm. This system was found to give compact spots for paracetamol (Rf value = 0.62 0.03), diclofenac potassium (0.75 0.02), and famotidine (0.17 0.03). The linear regression analysis data for the calibration plots showed a good linear relationship over the concentration range of 16259750 ng/spot for paracetamol, 2501500 ng/spot for diclofenac potassium, and 100600 ng/spot for famotidine. The method was validated for precision, robustness, and recovery according to International Conference on Harmonization guidelines. No chromatographic interference from the tablet excipients was found. Statistical analysis showed that the method was repeatable and selective for the simultaneous quantitation of the three drugs in tablet formulation and for routine quality control of raw materials of the drugs.
Development and Validation of a Reversed-Phase High-Performance Thin-Layer ChromatographyDensitometric Method for Determination of Atorvastatin Calcium in Bulk Drug and Tablets
Authors: Shirkhedkar Atul A , Surana Sanjay J
Published: February 2020

Atorvastatin calcium is a synthetic HMGCoA reductase inhibitor that is used as a cholesterol-lowering agent. A simple, sensitive, selective, and precise RP-HPTLCdensitometric determination of atorvastatin calcium both as bulk drug and from pharmaceutical formulation was developed and validated according to International Conference on Harmonization guidelines. The method used aluminum sheets precoated with silica gel 60 RP18F254s as the stationary phase, and the mobile phase consisted of methanolwater (3.5 + 1.5, v/v). The system gave a compact band for atorvastatin calcium with an Rf value of 0.62 0.02. Densitometric quantification was carried out at 246 nm. The linear regression analysis data for the calibration plots showed a good linear relationship with r = 0.9992 in the working concentration range of 100-800 ng/band. The method was validated for precision, accuracy, ruggedness, robustness, specificity, recovery, LOD, and LOQ. The LOD and LOQ were 6 and 18 ng, respectively. The drug underwent hydrolysis when subjected to acidic conditions and was found to be stable under alkali, oxidation, dry heat, and photodegradation conditions. Statistical analysis proved that the developed RP-HPTLCdensitometry method is reproducible and selective and that it can be applied for identification and quantitative determination of atorvastatin calcium in bulk drug and tablet formulation.
Development and Validation of an HPTLC Method for Determination of Olanzapine in Formulations
Authors: Patel Rashmin B , Patel Mrunali R , Bhatt Kashyap K , Patel Bharat G
Published: February 2020

A new, simple, and rapid HPTLC method was developed and validated for quantitative determination of olanzapine on silica gel 60F254 layers using methanolethyl acetate (8.0 + 2.0, v/v) as the mobile phase. Olanzapine was quantified by densitometric analysis at 285 nm. The method was found to give compact bands for the drug (Rf = 0.35 0.02). The linear regression analysis data for the calibration plots showed a good linear relationship with r2 = 0.9997 in the concentration range of 100600 ng/band. The method was validated for precision, recovery, repeatability, and robustness as per the International Conference on Harmonization guidelines. The LOD was found to be 23.90 ng/band, and the LOQ was 91.04 ng/band. Statistical analysis of the data showed that the method is precise, accurate, reproducible, and selective for the analysis of olanzapine. The method was successfully used for the determination of equilibrium solubility and quantification of olanzapine as a bulk drug, in a commercially available preparation, and in in-house developed mucoadhesive microemulsion formulations and solution.
Quantification of Phospholipids in Infant Formula and Growing Up Milk by High-Performance Liquid Chromatography with Evaporative Light Scattering Detector
Authors: Braun Marcel , Flück Brigitte , Cotting Claudia , Monard Florence , Giuffrida Francesca
Published: February 2020

Phospholipids (PLs) are well known for their excellent emulsifier properties and more recently for their biological functions, such as cell signing, brain development, immune function, heart health, and cancer prevention, besides their physiological role in membrane composition. In dairy products, PLs represent 0.21 of milk fat. The milk PLs comprise phosphatidylcholine (PC), phosphatidylethanolamine (PE), and sphingomyelin (SPH) as the major compounds; phosphatidylinositol and phosphatidylserine are minor PLs. A new generation of dairy products claiming PL family content, such as SPH, is being produced; therefore, a validated method for quantifying PL families in dairy products is needed. In this study, an HPLC-evaporative light scattering detector method to quantify the most abundant milk PL families, i.e., PC, PE, and SPH, in infant formula and growing up milk was developed and validated.
Sugar and Nonvolatile Acid Composition of Blackberries
Authors: Fan-Chiang Ho-Ju , Wrolstad Ronald E
Published: February 2020

Sugar and nonvolatile acid analyses were conducted on 52 samples of blackberries (Rubus spp), the objective being to develop a compositional database for evaluating authenticity and quality. Brix ranged from 6.88 to 16.83, with a mean of 10.82. Titratable acidity ranged from 0.52 to 2.24 g citric acid/100 mL, with a mean of 1.35. Sucrose levels (range, 012.9; mean, 4.6) were highly variable. The overall glucose:fructose ratio ranged from 0.81 to 1.17, with a mean of 1.01. Malic, isocitric, lactoisocitric, citric, shikimic, and fumaric acids were identified, with succinic acid being present in some samples. Malic acid ranged from 5.2 to 35.3 of total acids (87.5603 mg/100 g), with a mean of 16.4 (280 mg/100 g). Isocitric acid ranged from 4.7 to 71.6, with a mean of 34.7 (599 mg/100 g), and lactoisocitric acid ranged from 3.4 to 32.6 with a mean of 17.3 (293 mg/100 g). Citric acid ranged from 1.3 to 80.2, with a mean of 31.6 (572 mg/100 g). Shikimic, fumaric, and succinic were present in trace quantities. Two patterns of nonvolatile acid compositions were evident. Ten commercial blackberry juice samples were analyzed, and it was possible to determine whether they were Marion, Evergreen, or a mixture of the two from their acid profiles.
Reversed-Phase HPLC Analysis of Levetiracetam in Tablets Using Monolithic and Conventional C18 Silica Columns
Authors: Can Nafiz O , Arli Goksel
Published: February 2020

Development and validation of an RP-HPLC method for determination of levetiracetam in pharmaceutical tablets is described. The separation and quantification of levetiracetam and caffeine (internal standard) were performed using a single analytical procedure with two different types of stationary phases, conventional Phenomenex Gemini C18 (100 4.6 mm, 5 m) and Merck Chromolith Performance RP18e (100 4.6 mm, macropore size 2 mm, micropore size 13 nm) monolithic silica. Five-microliter aliquots of samples were injected into the system and eluted using wateracetonitrile (90 + 10, v/v) mobile phase pumped at the rate of 1 mL/min. The analyte peaks were detected at 200 nm using a diode array detector with adequate resolution. Validation studies were performed using the method recommended by the International Conference on Harmonization, the U.S. Pharmacopeia, and AOAC INTERNATIONAL, which includes accuracy, precision, range, limits, robustness, and system suitability parameters. Levetiracetam and caffeine were detected in about 7 min using the conventional column, whereas less than 5 min was required when the monolithic column was used. Calibration plots had r values close to unity in the range of 0.88.0 g/mL. Assay of levetiracetam in a tablet formulation was demonstrated as an application to real samples.
Development of an Ion-Pair Reversed-Phase HPLC Method with Indirect UV Detection for Determination of Phosphates and Phosphites as Impurities in Sodium Risedronate
Authors: Brezovska Katerina , Dimitrovska Aneta , Kitanovski Zoran , Petrusevska Jelena , Ribarska Jasmina Tonic , Jolevska Suzana Trajkovic
Published: February 2020

A method based on RP-HPLC with indirect UV detection was developed for the determination of phosphates and phosphites as impurities in sodium risedronate. RP separation of the phosphates and phosphites was achieved by adding tetrabutylammonium hydroxide as an ion-pairing agent in the mobile phase. Potassium hydrogen phthalate was added to the mobile phase as an ionic chromophore in order to obtain high background absorption of the mobile phase. Separation was performed on a C18 column using a mixture of pH 8.2 buffer (containing 0.5 mM tetrabutylammonium hydroxide and 1 mM phthalate) and acetonitrile (95 + 5, v/v) as the mobile phase, with indirect UV detection at 248 nm. The validation of the method included determination of specificity/selectivity, linearity, LOD, LOQ, accuracy, precision, and robustness. The LOD was 0.86 g/mL for phosphates and 0.76 g/mL for phosphites. The LOQ was 2.60 g/mL for phosphates and 2.29 g/mL for phosphites. The developed method is suitable for quantitative determination of phosphates and phosphites as impurities in QC of sodium risedronate.
Analysis of Salazinic, Norstictic, and Usnic Acids in Xanthoparmelia chlorochroa by Ultra-Performance Liquid Chromatography/Tandem Mass Spectrometry
Authors: Dailey Rebecca , Siemion Roger , Raisbeck Merl , Jesse Chris
Published: February 2020

The lichen species Xanthoparmelia chlorochroa is toxic when consumed by domestic sheep, cattle, and Rocky Mountain elk. Clinical signs exhibited by poisoned animals include red urine, ataxia, and muscular weakness that rapidly progresses to recumbency. Elk are unable to recover once becoming recumbent; however, most affected cattle can recover if offered suitable feed shortly following the onset of signs. At present, the pathogenesis and specific toxin(s) are unknown. As part of an effort to elucidate the proximate toxin, a method using ultra-performance LC coupled to MS/MS with negative-ion electrospray ionization has been developed to compare salazinic, norstictic, and usnic acid concentrations in X. chlorochroa collected from locales associated with lichen poisonings. Compounds were extracted from lichen samples with acetone and sonication. The stationary phase was a Waters Acquity UPLCTM BEH C18 (50 2.1 mm; 1.7 m particle size) column. The mobile phase consisted of an acetonitrilewater gradient. The precision of the method was confirmed by an SD below 0.4 (n = 9) for triplicate samples. LOD values were 200, 100, and 50 ng/mL for salazinic, norstictic, and usnic acids, respectively.
A Practical Approach for Linearity Assessment of Calibration Curves Under the International Union of Pure and Applied Chemistry (IUPAC) Guidelines for an In-House Validation of Method of Analysis
Authors: Sanagi M Marsin , Nasir Zalilah , Ling Susie Lu , Hermawan Dadan , Ibrahim Wan Aini Wan , Naim Ahmedy Abu
Published: February 2020

Linearity assessment as required in method validation has always been subject to different interpretations and definitions by various guidelines and protocols. However, there are very limited applicable implementation procedures that can be followed by a laboratory chemist in assessing linearity. Thus, this work proposes a simple method for linearity assessment in method validation by a regression analysis that covers experimental design, estimation of the parameters, outlier treatment, and evaluation of the assumptions according to the International Union of Pure and Applied Chemistry guidelines. The suitability of this procedure was demonstrated by its application to an in-house validation for the determination of plasticizers in plastic food packaging by GC.
Determination of Bithionol, Bromophen, Nitroxynil, Oxyclozanide, and Tribromsalan in Milk with Liquid Chromatography Coupled with Tandem Mass Spectrometry
Authors: Sakamoto Miho , Takeba Kazue , Sasamoto Takeo , Kusano Tomoko , Hayashi Hiroshi , Kanai Setsuko , Kanda Maki , Nagayama Toshihiro
Published: February 2020

LC/MS/MS was developed to determine the residues of bithionol (BTN), bromofen (BMF), nitroxynil (NTX), oxyclozanide (OCZ), and tribromsalan (TBS) in milk. Samples were extracted with ethyl acetate and cleaned up by liquidliquid separation with acetonitrile and n-hexane. The compounds were determined by RP-LC using a C18 column with 0.1 formic acidmethanol. Mass spectral acquisition was performed in the negative mode by applying selected-reaction monitoring. The method was validated in milk spiked with these compounds at 5600 g/kg; average recoveries were in the range 83.897.1, with RSD values of 1.48.0. The interassay RSDs were less than 11. The LODs of these compounds in milk were 0.1 g/kg. The method was applied to 24 raw milk samples. The concentration of these compounds in all samples was lower than the Japanese maximum residue limits. The method is rapid, sensitive, and specific for monitoring residues of BTN, BMF, NTX, OCZ, and TBS in milk.
Use of High-Performance Thin Layer Chromatography by the American Herbal Pharmacopoeia
Authors: Upton Roy T
Published: February 2020

TLC characterizations are among the key identity tests in most pharmacopoeial monographs. Pharmacopoeial standards are typically used by industry as a basis for meeting QC requirements and current good manufacturing practices (cGMPs). TLC is a relatively low-cost, highly versatile tool for developing specifications for raw materials, as well as for the various preparations for which pharmacopoeial standards are created. In addition to its use in the development of identity tests, TLC is a valuable tool for screening plant samples that pharmacopoeias must review in the development of monographs and botanical reference materials (BRMs). Specifically, HPTLC is the ideal TLC technique for these purposes because of its increased accuracy, reproducibility, and ability to document the results, compared with standard TLC. Because of this, HPTLC technologies are also the most appropriate TLC technique for conformity with GMPs. This article highlights the manner in which HPTLC is used by the American Herbal Pharmacopoeia (AHP) in the development of AHP monograph identity standards, the identification of adulterating species, and the development of AHP-verified BRMs.
Fingerprint Analysis and the Application of HPTLC to the Determination of Identity and Quality of Botanicals, from an Industry Perspective
Authors: Sudberg Sidney , Sudberg Elan M , Terrazas Jennifer , Sudberg Sandra , Patel Kirtal , Pineda Jose , Fine Bryan
Published: February 2020

There are many examples of botanical identity methodology currently in use today; all have associated levels of confidence and, hence, application for their intended purpose. This paper addresses the application of some of these methods to identify and qualify commonly traded herbs. The relevance of botanical identity and quality within the dietary supplement industry has seen a dramatic increase in the last several years, and now with the current Good Manufacturing Practices (cGMPs) recently published and the requirement for 100% identity testing, there is an increased need for reliable methods of analysis and subsequent interpretation of the results or data derived from these methods, given the diversity of samples and matrixes seen on a daily basis in a commercial setting. Example applications of methods described herein illustrate how one can confidently and effectively satisfy the 100% identity rule of the cGMPs to identify crude raw materials and their powdered or liquid extracts as well as identify a majority of such ingredients in finished products with diverse matrixes. The three techniques discussed in this paper are viewed from a qualitative perspective with primary emphasis on the application of HPTLC to a variety of complex samples with varying degrees of difficulty of analysis and interpretation. The three methods discussed are (1) HPTLC, for its fingerprint/pattern recognition capabilities as well as marker compound analysis; (2) microscopy, for identification/recognition of unique cellular, anatomic features and characteristics of the target plant or adulterants; and (3) HPLC for marker compound identification, quantitation, and/or other fingerprint qualities it may contribute to the data set. To support the applications and methods described herein, this paper presents four key approaches to assist in the interpretation of the data collected in order to correctly determine the identity of botanicals and their powdered extracts: the marker approach, the multicomponent-based approach, the pattern approach, and the multipattern approach, and how these approaches can be used with a vanguard/rearguard strategy in analytical testing as well as forming a basis for the functional fingerprint and the concept of phytoequivalence.