Determination of Vitamin B12 in Food Products by Liquid Chromatography/UV Detection with Immunoaffinity Extraction: Single-Laboratory Validation

Abstract A fast and simple method to determine vitamin B12 in foods is presented. The method allows, in addition to the determination of added cyanocobalamin, the determination of natural vitamin B12 forms, making it also applicable to nonfortified products, especially those that are milk-based. Vitamin B12 is extracted in sodium acetate buffer in the presence of sodium cyanide (100C, 30 min). After purification and concentration with an immunoaffinity column, vitamin B12 is determined by liquid chromatography with UV detection (361 nm). The method has been validated in analyses of a large range of products: milk- and soy-based infant formulas, cereals, cocoa beverages, health care products, and polyvitamin premixes. The method showed appropriate performance characteristics: linear response over a large range of concentrations, recovery rates of 100.8 7.5 (average standard deviation), relative standard deviation of repeatability, RSDr, of 2.1, and intermediate reproducibility, RSDiR, of 4.3. Limits of detection and quantitation were 0.10 and 0.30 g/100 g, respectively, and correlation with the reference microbiological assay was good (R2= 0.9442). The proposed method is suitable for the routine determination of vitamin B12 in fortified foods, as well as in nonfortified dairy products. It can be used as a faster, more selective, and more precise alternative to the classical microbiological determination.

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Determination of Dislodgeable Residues of N-4-Chlorobenzoyl-N’-Benzoyl-N’-tert-Butylhydrazine (RH-0345) from Grass by Liquid Chromatography

Journal of AOAC International ◽

10.1093/jaoac/80.2.418 ◽

1997 ◽

Vol 80 (2) ◽

pp. 418-425

Author(s):

Stephen S Skorczynskis

Keyword(s):

Liquid Chromatography ◽

Standard Deviation ◽

Surface Area ◽

Methylene Chloride ◽

Threshold Value ◽

Total Surface Area ◽

Deionized Water ◽

Uv Detection ◽

Simple Method

Abstract A simple method for determining chemically and physically dislodgeable residues of N-4-chloroben-zoyl-N′-benzoyl- N′-tert-butylhydrazine (RH-0345) from grass is described. To determine chemical dis- lodgeability, a sample of fresh grass leaves is washed with deionized water containing a small amount of the wetting agent Aerosol OT-75. Quantitation of residues removed from the grass is based on the total surface area of grass analyzed. Physical dislodgeability is measured by sampling an area of turf for 1 min. Sampling is accomplished by a person wearing a dosimeter made from a common athletic sock. Grass-stained socks are extracted with 75% acetonitrile in deionized water, and the extracts are diluted with water. Quantitation of physically dislodgeable residues is based on the total RH-0345 residue adsorbed on the sock and is expressed as ng RH-0345/cm2 of sock in contact with the grass. For amounts of dislodgeable residues greater than the threshold value of 250 ng/cm2 for grass and 100 ng/cm2 for socks, extracts are analyzed directly by liquid chromatography (LC) using a C8 column and UV detection at 260 nm. For samples with significantly lower amounts of dislodgeable residues, diluted extracts are partitioned with methylene chloride. The methylene chloride containing the RH-0345 residues is evaporated just to dryness, and the residues are reconstituted in 30% acetonitrile in water and then analyzed by LC. The validated sensitivity of the method is 0.5 ng/cm2 for grass and 50 ng/cm2 for cotton socks. Grass was fortified at 0.5–10 000 ng/cm2. Average RH-0345 recovery from grass was 94 ± 10% standard deviation (SD). Apparent RH-0345 residues in control grass were 0.0558 ng/cm2. Socks were fortified at 50–40 000 ng/cm2. Average RH-0345 recovery from socks was 98 ± 7% SD. Apparent RH-0345 residues in control socks were 6.13 ng/cm2.

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Determination of Nitrate in Water by HPLC

Applied Mechanics and Materials ◽

10.4028/www.scientific.net/amm.448-453.406 ◽

2013 ◽

Vol 448-453 ◽

pp. 406-408

Author(s):

Jing Liu ◽

Xiao Na Ji ◽

Qing Kai Ren ◽

Sheng Shu Ai ◽

Li Jun Wan ◽

...

Keyword(s):

High Performance Liquid Chromatography ◽

Liquid Chromatography ◽

Standard Deviation ◽

Relative Standard Deviation ◽

Recovery Rate ◽

High Performance ◽

Separation Efficiency ◽

Fast Analysis ◽

Relative Standard

We established a method fordetermination of nitrate in water by High Performance Liquid Chromatography(HPLC). The sample was analysed by HPLC-ADA and was quantitated by externalstandard method after being simply processed. This methd has the advantages ofhigh separation efficiency and fast analysis. The experiment result showed thatthe linearly dependent coefficient was0.994, the recovery rate was between 98.7%～105.7%,the relative standard deviation（RSD）wasless than 2.1 %, and the lowest detectable limit is 0.01ng (S/N=1.6).

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Gas-Liquid Chromatography of Trichlorfon in Soluble Powder Formulations

Journal of AOAC INTERNATIONAL ◽

10.1093/jaoac/57.5.1128 ◽

1974 ◽

Vol 57 (5) ◽

pp. 1128-1131

Author(s):

Phil B Bowman ◽

Peter W Dame

Keyword(s):

Liquid Chromatography ◽

Standard Deviation ◽

Relative Standard Deviation ◽

Trimethylsilyl Ether ◽

Gas Liquid Chromatography ◽

Mass Spectral ◽

Powder Formulation ◽

Relative Standard ◽

Quantitative Recovery

Abstract A procedure is described for the determination of trichlorfon in a soluble powder formulation by gas-liquid chromatography. Silylation prevents on-column degradation of trichlorfon to dichlorvos. The procedure provides quantitative recovery from the formulation as demonstrated by a spiking study. A relative standard deviation of less than 2% was obtained for 6 replicate assays of a single lot of formulation. The mass spectral fragmentations of trichlorfon and trichlorfon-trimethylsilyl ether are described.

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Determination of Patulin in Apple Juice by Liquid Chromatography

Journal of AOAC International ◽

10.1093/jaoac/89.1.139 ◽

2006 ◽

Vol 89 (1) ◽

pp. 139-143 ◽

Cited By ~ 20

Author(s):

Maria Helena Iha ◽

Myrna Sabino

Keyword(s):

Liquid Chromatography ◽

Standard Deviation ◽

Mobile Phase ◽

Apple Juice ◽

Array Detector ◽

Diode Array ◽

Diode Array Detector ◽

Relative Standard ◽

Precision Study

Abstract A method was developed and validated in-house for the determination of patulin (PAT), a toxic mold metabolite, in apple juice. The sample was extracted with ethyl acetatehexane and analyzed by liquid chromatography equipped with a C18 column and diode array detector. The mobile phase used for the quantification was waterethanol, at a flow rate of 0.5 mL/min. The method showed a mean recovery of 84.8%, the relative standard deviation obtained in the precision study was <7.7%, the quantification and detection limits were 7 and 3 μg/L, respectively, and the linear range for PAT in apple juice was 2.6650 μg/L. The ruggedness was evaluated by an intralaboratory experiment, in which 5 factors were studied, and only one was found to influence the observed results. The developed method is fast, practical, and simple; the solvents (except hexane) and reagents used were nontoxic. The results of the validation confirmed the efficiency of the method, which is sensitive enough to be used in studies required to quantify PAT in apple juice.

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Validation of HPLC Method for Determination of Histamine in Human Immunoglobulin Formulations

Journal of AOAC International ◽

10.1093/jaoacint/qsaa017 ◽

2020 ◽

Vol 103 (5) ◽

pp. 1223-1229

Author(s):

Michikazu Tanio ◽

Toru Nakamura ◽

Hideki Kusunoki ◽

Kyohei Ideguchi ◽

Kazuyuki Nakashima ◽

...

Keyword(s):

High Performance Liquid Chromatography ◽

Liquid Chromatography ◽

Standard Deviation ◽

High Performance ◽

Gradient Elution ◽

Hplc Method ◽

Human Immunoglobulin ◽

Histamine Dihydrochloride ◽

Relative Standard

Abstract Background Histamine fixed-immunoglobulin formulations, which consisted of 0.15 µg of histamine dihydrochloride and 12 mg of human immunoglobulin in a vial, are used for anti-allergic treatments, and controlling the amounts of histamine in the formulations is essential to avoid histamine intoxication. Objective A high-performance liquid chromatography (HPLC) method for determination of histamine contents of the formulations was established and validated. Methods Histamine extracted from the formulation was labeled with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate and was analyzed by gradient elution HPLC with UV detection at 260 nm. Results The method showed linearity in the range 0.8–2.4 µM (R > 0.999), accuracy (100.1–105.8% recovery), and precision (relative standard deviation ≤ 1.93%). The validated method was applied for five lots of the pharmaceutical, and their histamine contents were determined to be 0.149–0.155 µg/vial. Conclusions These results indicated that the validated method is useful to control amounts of histamine in biopharmaceutical products. Highlights The HPLC method was developed for quantitative determination of histamine content of the histamine fixed-immunoglobulin formulations.

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Immunoaffinity Column Cleanup with Liquid Chromatography Using Post-Column Bromination for Determination of Aflatoxins in Hazelnut Paste: Interlaboratory Study

Journal of AOAC International ◽

10.1093/jaoac/88.2.526 ◽

2005 ◽

Vol 88 (2) ◽

pp. 526-535 ◽

Cited By ~ 18

Author(s):

Hamide Z Senyuva ◽

John Gilbert

Keyword(s):

Liquid Chromatography ◽

Standard Deviation ◽

Relative Standard Deviation ◽

Aflatoxin B1 ◽

The United States ◽

Interlaboratory Study ◽

Immunoaffinity Column ◽

Test Portion ◽

Relative Standard

Abstract An interlaboratory study was conducted to evaluate the effectiveness of an immunoaffinity column cleanup liquid chromatography (LC) method for determination of aflatoxin B1 and total aflatoxins in hazelnut paste at European regulatory limits. The test portion was extracted with methanol–water (6 + 4). The extract was filtered, diluted with phosphate-buffered saline (PBS) solution to a specified solvent concentration, and applied to an immunoaffinity column containing antibodies specific to aflatoxins. The aflatoxins were removed from the immunoaffinity column with methanol, and then quantified by reversed-phase LC with post-column derivatization (PCD) involving bromination. The PCD was achieved with electrochemically generated bromine (Kobra Cell®) followed by fluorescence detection (except for one participant who used pyridinum hydrobromide perbromide for bromination). Hazelnut paste, both naturally contaminated with aflatoxins and blank (<0.1 ng/g) for spiking by participants with aflatoxins, was sent to 14 collaborators in Belgium, The Netherlands, Spain, Turkey, the United Kingdom, and the United States. Test portions were spiked at levels of 4.0 and 10.0 ng/g for total aflatoxins by participants using supplied total aflatoxins standards. Recoveries for total aflatoxins and aflatoxin B1 averaged from 86 to 89%. Based on results for naturally contaminated samples (blind duplicates at 3 levels ranging from 4.0 to 11.8 ng/g total aflatoxins), the relative standard deviation for repeatability (RSDr) ranged from 2.3 to 3.4% for total aflatoxins and from 2.2 to 3.2% for aflatoxin B1. The relative standard deviation for reproducibility (RSDR) ranged from 6.1 to 7.0% for total aflatoxins and from 7.3 to 7.8% for aflatoxin B1. The method showed exceptionally good within-laboratory and between-laboratory precision for hazelnut paste, as evidenced by HORRAT values, which in all cases were significantly below target levels, the low levels of determination for both aflatoxin B1 and total aflatoxins.

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Determination of Cholecalciferol (Vitamin D3) in Selected Foods by Liquid Chromatography: NMKL Collaborative Study

Journal of AOAC International ◽

10.1093/jaoac/86.2.400 ◽

2003 ◽

Vol 86 (2) ◽

pp. 400-406 ◽

Cited By ~ 20

Author(s):

Anders Staffas ◽

Arne Nyman ◽

K Ask ◽

E Hermansson ◽

J S Jacobsen ◽

...

Keyword(s):

Liquid Chromatography ◽

Standard Deviation ◽

Fish Oil ◽

Vitamin D3 ◽

Collaborative Study ◽

The Other ◽

Vitamin D2 ◽

Cooking Oil ◽

Relative Standard

Abstract Results are presented from an NMKL (Nordic Committee on Food Analysis) collaborative study of a method for the determination of cholecalciferol (vitamin D3) in foods. The method is based on the addition of an internal standard (vitamin D2), followed by saponification and extraction with n-heptane. The fraction that contains vitamin D2/D3 is separated by preparative normal-phase liquid chromatography (LC), and the analytes are determined by reversed-phase LC with UV detection at 265 nm. The method was tested by 8 participating laboratories. In this study 6 different matrixes were analyzed for cholecalciferol content: milk, liquid infant formula (gruel), cooking oil, margarine, infant formula, and fish oil. The contents varied from 0.4 to 12 μg/100 g. Three matrixes (milk, gruel, and margarine) were fortified with vitamin D3. In the other matrixes, vitamin D3 was added at 3 different levels at the Swedish National Food Administration. The milk was analyzed as a blind duplicate, whereas the other matrixes were analyzed as split-level pairs. The recoveries from the samples with vitamin D3 added varied from 93 to 102%. The repeatability relative standard deviation (RSDr) values for accepted results varied between 2.2% (fish oil) and 7.4% (cooking oil), whereas the reproducibility relative standard deviation (RSDR) values varied between 6.8% (margarine) and 24% (cooking oil).

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Determination of p-Toluenesulfonamide in Ice Cream by Combination of Continuous Flow and Liquid Chromatography: Summary of Collaborative Study

Journal of AOAC International ◽

10.1093/jaoac/77.3.672 ◽

1994 ◽

Vol 77 (3) ◽

pp. 672-674 ◽

Cited By ~ 3

Author(s):

Paul R Beuaars ◽

Remmelt Van Dijk ◽

Arie Brands

Keyword(s):

Liquid Chromatography ◽

Standard Deviation ◽

Relative Standard Deviation ◽

Continuous Flow ◽

Collaborative Study ◽

Ice Cream ◽

Relative Standard ◽

On Line ◽

Positive Results

Abstract A collaborative study of the determination of p-tolu-enesulfonamide (p-TSA) in ice cream by a combination of continuous flow and on-line liquid chromatography was conducted. Seven ice cream samples containing 0-6.35 mg p-TSA/kg at 4 levels (1 blank and 3 pairs of split level samples) were analyzed by 11 laboratories. For all samples analyzed, the repeatability relative standard deviation varied from 2.08 to 3.67%, whereas the reproducibility relative standard deviation ranged from 7.79 to 11.68%. The average p-TSA values for the split levels 1,2, and 3 were 0.55,1.02, and 4.44 mg p-TSA/kg, respectively, with mean recoveries ranging from 76 to 79% (overall recovery range for all levels, 63-101 %). No false positive results were reported for the blank sample, and no interference was encountered by the presence of vanillin in samples. The method has been adopted first action by AOAC INTERNATIONAL.

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Determination of Clopidol Residues in Chicken Tissues by Liquid Chromatography: Collaborative Study

Journal of AOAC International ◽

10.1093/jaoac/86.4.685 ◽

2003 ◽

Vol 86 (4) ◽

pp. 685-693 ◽

Cited By ~ 3

Author(s):

Guo-Fang Pang ◽

Yan-Zhong Cao ◽

Chun-Lin Fan ◽

Jin-Jie Zhang ◽

Xue-Min Li ◽

...

Keyword(s):

Liquid Chromatography ◽

Standard Deviation ◽

Relative Standard Deviation ◽

Solid Phase ◽

Collaborative Study ◽

Reversed Phase ◽

Method Performance ◽

Chicken Muscle ◽

Relative Standard

Abstract Eighteen laboratories participated in a collaborative study on the determination of clopidol residues in chicken muscle tissues by liquid chromatography. Of these, results from 16 laboratories which rigorously followed the method were subjected to statistical analysis. The method performance was assessed by all participants using 14 samples of chicken muscle fortified at concentrations ranging from 0.1 to 5.0 mg/kg. In addition, 9 participants each reported results for 6 clopidol-incurred samples in chicken muscle. Test portions were extracted with acetonitrile, and the extracts were purified with alumina and anion exchange resin solid-phase extraction cartridges in sequence. Clopidol was separated by reversed-phase liquid chromatography and quantified at 270 nm. Average recoveries ranged from 81.8 to 85.4%, reproducibility relative standard deviation (RSDR) ranged from 11.9 to 22.6%, and repeatability relative standard deviation (RSDr) ranged from 9.9 to 15.1%. For clopidol-incurred samples at concentrations of 0.100–0.687 mg/kg, the mean determination value range was 0.099–0.659 mg/kg; RSDR was 12.6–19.8%, RSDr was 3.1–8.5%; and HORRAT values were 0.7–1.1. The accuracy and precision of the method are in conformity with the requirements specified by AOAC INTERNATIONAL. The method was adopted Official First Action in April 2003.

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Simultaneous Determination of Free Carnitine and Total Choline by Liquid Chromatography/Mass Spectrometry in Infant Formula and Health-Care Products: Single-Laboratory Validation

Journal of AOAC International ◽

10.1093/jaoac/91.4.777 ◽

2008 ◽

Vol 91 (4) ◽

pp. 777-785 ◽

Cited By ~ 11

Author(s):

Pierre Andrieux ◽

Tamara Kilinc ◽

Christian Perrin ◽

Esther Campos-Giménez

Keyword(s):

Health Care ◽

Standard Deviation ◽

Simultaneous Determination ◽

Relative Standard Deviation ◽

Infant Formula ◽

Free Carnitine ◽

Official Method ◽

Relative Standard ◽

Single Laboratory

Abstract A single-laboratory validation study was conducted for a liquid chromatographic/mass spectrometric (LC/MS) method for the simultaneous determination of the free carnitine and total choline in milk-based infant formula and health-care products. The sample preparation used for both carnitine and choline was adapted from AOAC Official Method 999.14, with an acidic and enzymatic hydrolysis of esterified forms of choline. Carnitine and choline were quantified by ion-pair chromatography with single-quadrupole MS detection, using their respective deuterated internal standards. The repeatability relative standard deviation was 2.5 and 2.1, respectively, for carnitine and choline. The intermediate reproducibility relative standard deviation was <4.7 and 2.4, respectively, for carnitine and choline. The ranges of the average product-specific recoveries were 9298 and 94103, respectively, for carnitine and choline. Choline concentration determined in infant formula reference material SRM 1846 was in agreement with the reference value. The proposed method was compared with the enzymatic methods for a range of products; good correlation (r = 0.99) was obtained, although a significant bias was observed for both analytes. The method, with a short chromatographic run time (7 min), is convenient for routine analysis to enhance analytical throughput and is a good alternative to enzymatic assays.

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