Carn-06_Book_Final

Eurofins Steins A/S Carnitine/Choline (Carn-06:

Lars T. Tanderup Chemist M.Sc.

Eurofins Steins Laboratory A/S Vitamin Competence Centre 6600 Vejen, Denmark 2013-06-28

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Determination of Carnitine in Infant Formula, Adult/Pediatric Nutritional Formula, food and feed ESI LC-MS/MS (applicable to the determination of free and total carnitine in Food/feed, infant formula, adult nutritionals and nutritional supplement.) A. Principle Carnitine and esters of this are extracted by methanol and water. Free carnitine is determined on the extract. Total carnitine is measured after alkaline hydrolysis at elevated temperature of the extract, thereby converting the bound forms in to free carnitine. The resulting free Carnitine is quantified by isotope dilution LC-MSMS. B. Apparatus ( a ) Column. - Synergi 2,5μ polar-RP 100 x 3.00 mm (Phenomenex), or equivalent. ( b ) Liquid chromatograph. - Thermo Dionex Ultimate 3000 RS 600bar HPLC system, or equivalent. ( c ) Detector. - AB Sciex Q-Trap 3200 mass spectrometer, or equivalent. ( d ) Data system. – Analyst 1.5.1 and Dionex Chromatography MS link 2.10.0.2906 ( e ) Balance.—Readable to at least 0.0001 g (Satorius, or equivalent). ( f ) Balance.—Readable to at least 0.001 g (Kern, or equivalent). ( g ) Incubator. – Capable of 37 ± 5°C ( h ) Centrifuge. ( i ) Shaking table. (Inside Incubator) ( j ) Oven. - Capable of 102 ± 2°C ( k ) Centrifuge tubes.—plastic , 50 mL with screw cap. ( l ) Extraction vessels. – conical flasks, 250 ml with stoppers. ( m ) Digestion vessels. Conical flasks, 500 ml with stoppers. ( n ) Volumetric flasks. 50, 100 and 500 ml and 1 L C. Reagents ( a ) Purified Water, USP; ≥18 Mohm/cm. ( b ) Methanol. – HPLC-grade (for sample preparation) ( c ) Methanol. – LC-MS grade (for LC-MS/MS mobile phases preparation) ( d ) Ammonium formate. LC-MS grade, crystalline, ≥99.0% ( e ) Formic acid. LC-MS grade, ≥99.9% ( f ) Potasium hydroxide. – p.a., crystaline, ≥85% ( g ) Hydrochloric acid. –p.a., 37% D. Standards ( a ) L-Carnitine reference standard.—U.S. Pharmacopeia (Rockville, MD; Cat. No. 1359903). ( b ) L-Carnitine-d3 HCl (methyl-d3).—C/D/N Isotopes (Quebec, Canada; Cat. No. D-5069). ( o ) Syringe filters.— 0.45 μm. ( p ) Pipets.—5000 μL adjustable ( q ) Pipet tips.

( 1 ) Mobile phase A. – Measure 200 mL ammonium formate buffer into a 1 L volumetric flask and dilute to the mark with water. ( 2 ) Mobile phase B. – Methanol LC-MS grade ( c ) Potassium hydroxide solution. – 2 M Dissolve 56.1 g reagent grade KOH pellets in water in a 500 mL volumetric fl ask, and dilute to volume with water. Storage: plastic bottle, room temperature. Expiration: 1 year. ( d ) Hydrochloric acid.- 1.6 M. Measure 67.5 mL concentrated HCl into 500 mL volumetric flask and dilute to the mark with water. Standardization is not required. Expiration: 1 year. ( e ) L-Carnitine inner salt solution (approximately 4 mg/g). – Accurately weigh 400 mg L-carnitine reference standard as quickly as possible (because of hygroscopicity), and transfer to a 100 mL flask. Dissolve L-carnitine in water, and dilute to volume with water and accurately weigh the solution. The (approximately 400 µg/g). — Accurately weigh 10 g L- Carnitine inner salt solution into a 100 mL flask. Dilute to volume with water and accurately weigh the solution. ( g ) L-Carnitine Stock solution, (approximately 12 µg/g). — Accurately weigh 15 g L-Carnitine inner salt intermediate solution into a 500 mL flask. Dilute to volume with water and accurately weigh the solution. Dispense the solution into plastic centrifuge tubes, each receiving ≥10 mL. Storage: freezer, –20°C. Expiration: 1 year. ( h ) L-Carnitine-d 3 Stock solution, (approximately 80 µg/g). – Accurately weigh 50 mg L-carnitine-d 3 HCl (methyl-d 3 ) as quickly as possible, and transfer to a 500 mL flask. Dissolve L-carnitine-d 3 HCl (methyl-d 3 ) in water, and dilute to volume with water and accurately weigh the solution. Dispense the solution into plastic centrifuge tubes, each receiving ≥15 mL. Storage: freezer, –20°C. Expiration: 1 year. ( i ) ISTD standard solution (approximately 2.3 µg/g L- carnitine-d 3 ). – Accurately weigh 14 g L-Carnitine Stock solution into a 200 mL flask. Add 4 mL of 1.6 M Hydrochloric acid and 3 ml of 2 M Potassium hydroxide solution. Dilute to volume with water and accurately weigh the solution. This amount of internal standard is sufficient to 6 standards and 18 samples. ( j ) Standard solution (approximately 1.2 µg/g of L-carnitine and choline). – Accurately weigh 10 g L-Carnitine Stock solution and 10 g Choline Stock solution into a 100 mL flask. Dilute to volume with water and accurately weigh the solution. ( k ) Working standards. — Prepare fresh on day of analysis. ( 1 ) Level 1 calibration standard. - Accurately weigh 1 g standard solution and 4 g ISTD standard solution into a 100 mL flask. Dilute to volume with water and accurately weigh the solution. Cap and mix well. Transfer to an autosampler vial and cap. ( 2 ) Level 2 calibration standard. - Accurately weigh 3 g standard solution and 2 g ISTD standard solution into a 50 mL flask. Dilute to volume with water and accurately weigh the solution. Cap and mix well. Transfer to an autosampler vial and cap. ( 3 ) Level 3 calibration standard. - Accurately weigh 10 g standard solution and 2 g ISTD standard solution into a 50 mL flask. Dilute to volume with water and accurately weigh the solution. Cap and mix well. Transfer to an autosampler vial and cap. ( 4 ) Level 4 calibration standard. - Accurately weigh 20 g concentration is calculated on mass to mass basis. ( f ) L-Carnitine inner salt intermediate solution,

E. Solutions Preparation ( a ) Ammonium formate buffer. – 100mM, pH 3.0

Quantitatively transfer 1.12 ±0.01 g ammonium formate into a 1 L beaker add 800 ml water, dissolve, adjust pH to 3,0 with concentrated formic acid, transfer to volumetric flask and fill to volume with water. ( b ) Mobile phase. –

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standard solution and 2 g ISTD standard solution into a 50 mL flask. Dilute to volume with water and accurately weigh the solution. Cap and mix well. Transfer to an autosampler vial and cap. ( 5 ) Level 5 calibration standard. - Accurately weigh 26 g standard solution and 2 g ISTD standard solution into a 50 mL flask. Dilute to volume with water and accurately weigh the solution. Cap and mix well. Transfer to an autosampler vial and cap. ( 6 ) Level 6 calibration standard. - Accurately weigh 40 g standard solution and 2 g ISTD standard solution into a 50 mL flask. Dilute to volume with water and accurately weight the solution. Cap and mix well. Transfer to an autosampler vial and cap. ( 1 ) For dry blended/nonhomogeneous powder samples, transfer 25 g, accurately weighed to a 250 mL volumetric flask. Dissolve using warm distilled water ~40°C, cool, and make up to 250 g accurately weighed with distilled water. Transfer 10 - 15 g accurately weighed of reconstituted sample to the extraction vessels. ( 2 ) For wet blended homogeneous powder samples, transfer 2 - 3 g, accurately weighed directly to the extraction vessels. ( 3 ) For ready-to-feed samples or concentrated liquid products, transfer 5 to 10 g, accurately weighed of thoroughly agitated sample directly to the extraction vessel. ( 4 ) For dry food or animal feed samples, transfer 5 - 10 g, accurately weighed of sample directly to the extraction vessel. ( 5 ) For wet food or animal feed samples, transfer 10 - 15 g, accurately weighed of sample directly to the extraction vessel. ( 6 ) For nutritional supplements, transfer 1 – 1.5 g, accurately weighed of sample directly to the extraction vessel. ( c ) Ekstraction. – ( 1 ) For liquid samples no added water is necessary. For wet samples add 5 mL of water. For dry samples add 10 ml of water. ( 2 ) Add 100 ml of Methanol (HPLC-grade), and cap. ( 3 ) Mix by shaking until well dispersed. Place flask on shaking table at 37°C for 12 h. adjust shaking to obtain good mixing. ( 4 ) Cool to ambient temperature. ( 5 ) Accurately weigh the extraction vessel. ( 6 ) Transfer part of the extraction liquid to a 50 mL centrifuge tube, and cap. ( 7 ) Centrifuge the extraction liquid to obtain a clear liquid, ( 1 ) For high concentration samples a dilution step with water should be performed before addition of the internal standard. ( 2 ) Accurately weigh the digestion vessels. ( 3 ) Transfer 1 – 5 g accurately weighed, of the clear extraction liquid to the digestion vessels. ( 4 ) Add 10 g accurately weighed ISTD standard solution to the digestion vessels. ( 5 ) Dilute to 250 mL volume with water and accurately weight the solution. Cap and mix well. Transfer to an auto- sampler vial and cap. ( e ) Determination of Total carnitine and – ( 1 ) For high concentration samples a dilution step with water should be performed before addition of the internal standard.. (approximately 200 rpm for 10 min). ( d ) Determination of free carnitine - F. Sample preparation and Extraction ( a ) Sample weighing. – ( b ) Accurately weighed the extraction vessels.

( 2 ) Accurately weigh the digestion vessels. ( 3 ) Transfer 1 – 5 g accurately weighed, of the clear extraction liquid to the digestion vessels. ( 4 ) Add 10 g accurately weighed ISTD standard solution to the digestion vessels. ( 5 ) Add 3 mL of 2M Potassium hydroxide solution, and 47 mL of water, close with stopper. ( 6 ) Hydrolyse in oven at 102°C for 4 h. ( 7 ) Cool to ambient temperature. ( 8 ) Add 4 mL 1.6M Hydrochloric acid ( 9 ) Dilute to 250 mL volume with water and accurately weight the solution. Cap and mix well. Transfer to an autosampler vial and cap. G. Instrument operation conditions ( a ) LC. – Isocratic run 0-2 min. 98% A; 2% B. Flow 0,5 mL/min; injection volume, 2 μL; column temperature, 25°C; autosampler temperature, ambient. ( b ) MS/MS.—Ionization mode, positive-ion electrospray ionization (ESI+);Curtain gas, 30 psi;Collision gas, Med; Ionspray voltage , 5.5 kV; source temperature, 700°C; Ion Source Gas 1, 65 psi;Ion source gas 2, 65 psi. ( c ) Parameters for MS/MS measurement see table 1.

Table 1. Analyte

Q1 a

Q3 b

DP c

EP d CE e 5.0 19.0 5.0 25.0 5.0 19.0 5.0 25.0

CXP f

L-carnitine L-carnitine

162.2 103.1 36.0

4.0 4.0 4.0 4.0

162.2

85.1 36.0

L-Carnitine-d 3 L-Carnitine-d 3

165.2 103.1 36.0

165.2

85.1 36.0

a Q1 = Quadrupole mass filter 1. b Q3 = Quadrupole mass filter 3. c DP = Declustering potential. d EP = Entrance potential. e CE = Collision energy. f CXP = Collision cell exit potential.

( d ) Data acquisition was done in the multiple-reaction monitoring (MRM) mode. ( e ) Quantitation of L-carnitine.—The MRM chromatogram for L-carnitine is a sum of signals for the transitions m/z 162 > 103 and 162 > 85. Likewise, the MRM chromatogram for the ISTD is a sum of signals for the transitions m/z 165 > 103 and 165 > 85. Analyst automatically calculates a response ratio, which is defined as the L-carnitine peak area divided by the ISTD peak area. ( f ) UPLC analysis.—After verifying equilibration of the UPLC system, inject the working standards (L1 to L6) followed by a reagent blank, control sample, and sample extracts. ( g ) Inject working standards after approximately every 20 sample extraction, injected after the analysis of the last sample extract. Notes: Calibration curves must have a correlation coefficient r 2 of >0.990.

H. Calculation of results ( a ) Manual calculations.—

( 1 ) Plot each calibration standard response ratio versus its corresponding concentration to obtain calibration curves for L-carnitine. Apply a nonweighted linear regression to the data, and obtain an equation for the best-fit line.

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Where y is the response ratio and x is the concentration (ng/g). ( 2 ) Calculate the concentration in a final sample dilution, by interpolating from the standard curve: Interpolated concn., (ng/g) = (sample response ratio – b)/m ( 3 ) Calculate the concentration in a sample, based on the interpolated concentration:

Where: W sample

: mass of weighed sample (g).

F : dillution factor.

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Eurofins Steins A/S Validation Report: Carnitine quantitated by liquid chromatography and isotope dilu- tion mass spectroscopy.

Lars T. Tanderup Chemist M.Sc.

Eurofins Steins Laboratory A/S Vitamin Competence Centre 6600 Vejen, Denmark 2013-06-28

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Table of Contents:

Abbreviations and synonyms: .....................................................................................................................................1 Quality statement:........................................................................................................................................................1 Introduction:.................................................................................................................................................................1 Brief method description:.............................................................................................................................................2 Statistics: .....................................................................................................................................................................2 Matrices: ......................................................................................................................................................................2 Specificity: ...................................................................................................................................................................2 Linearity: ......................................................................................................................................................................3 Precision:.....................................................................................................................................................................3 Trueness:.....................................................................................................................................................................4 Measuring range:.........................................................................................................................................................6 Limit of detection / quantification: ................................................................................................................................6 Robustness:.................................................................................................................................................................6 Summary and conclusions: .........................................................................................................................................9 Appendix 1: Linearity .................................................................................................................................................10 Appendix 2: Precision................................................................................................................................................12 Appendix 3: Limit of detection / quantification ...........................................................................................................13 Appendix 4: Robustness. ..........................................................................................................................................14

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Abbreviations and synonyms:

Abbreviations: NMKL.................Nordisk Metodikk Komité for Næringsmidler ISO ....................International Organization for Standardization SOP...................Standard Operating Procedure ANAVA ..............Analysis of Variance LOD ...................Limit of Detection LOQ...................Limit of Quantitation n ........................Number of replicates LC......................Liquid Chromatography MS .....................Mass spectroscopy R 2 .......................Squared regression coefficient RSD%................Relative Standard Deviation in Percent CI .......................Confidence intervals S r .......................Repeatability (standard deviation within days) S R ......................Reproducibility (standard deviation between days) SRM ..................Certified reference material NIST ..................National Institute of Standards and Technology Synonyms: t-test .................. Student’s t -test This validation report is conducted in accordance to our internal instruction 211.01 “Validering af analysemetoder i Vitamincenter (in English - Validation of analytical methods in the Vitamin Centre” This instruction defines the experimental design used in the current validation, which is based on NMKL Procedure No. 4: Validation of chemical analytical methods (2009, 3 rd edition). The Eurofins Steins Laboratory in Vejen is accredited according to ISO 17025. Quality statement:

Introduction:

This method was developed in the vitamin centre. The extraction is based on an old internal Eurofins method for determination of choline.

Page 1 of 16

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Eurofins Lab. LTD., Wolverhampton: SOP CHROM/341-Choline in pet foods by ion chromatography with conductivity detection.

The quantification by LC-MS/MS is based on the analytical concepts of the follow- ing two articles.

STARKEY et AL. J.AOAC INT. 91,1,2008,130-142.Single-Laboratory Validation of a Liquid chromatographic/Tandem Mass Spectrometric Method for the Deter- mination of Free and Total Carnitine in Infant Formula and Raw Ingredients.

Brief method description:

The sample is shaken in a mixture of water and methanol thereby extracting car- nitine including their phosphorylated forms. An aliquot is treated with potassium hydroxide solution at elevated temperature for 4 hours thereby converting the phosphorylated forms to free carnitine. The mixture is neutralised and deuterered internal standard is added. Quantification is done by LC-MS.

Statistics:

In the current validation report, a p-value < 0.05 is considered statistically signifi- cant. Consequently, confidence intervals (CI) are calculated at a confidence level at 95 %. Paired t-testing is performed on normal distributed raw data. Data which is not within the normal distribution is clearly marked within the text.

Matrices:

This method can be used for quantification of free and/or total carnitine in the fol- lowing matrices:

 Food and feed.  Supplements, premixes and raw products.  Milk powder and nutritional formulas.  Animal feed / pet food (dry and wet).

Specificity:

The specificity has been evaluated by examining chromatograms of natural sam-

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ples of the above mentioned sample types. No interferences have been ob- served.

Linearity:

The Linearity has been evaluated twice for both compounds with a 9 points cali- bration. For the data and the result of the regression analysis performed in excel, see ap-

pendix 1. Carnitine:

There is seen a very significant correlation between concentration ratio and area ratio. R 2 =0,9998. The slope of the line is 0,9596 and the intercept with the y-axis is 0,0046. The calculated 95% confidence interval for the intercept includes 0. The calibration curve therefore goes through 0. For residuals plotted against the concentration ratio, it is seen that the points are randomly distributed around the x-axis (concentration ratio), indicating that the calibration curve for the concentrations included are linear. For the evaluation of the precision two types of samples have been used:  A milk powder (nutritional formula).  A pet food. (dry pet food) The repeatability (S r ) and reproducibility (S R ) have been evaluated with a variance analysis model. The data consists of 16 days of double replicate measurements. The dataset has been evaluated with ANAVA single factor (Excel 2010), see ap- pendix 2. Milk powder (mg/kg) Pet food (mg/kg) Carnitine Carnitine Reproducibility (S R ) 3,1% 3,3% Repeatability (S r ) 2,6% 1,9% F 1,865 5,188 F crit 2,352 2,352 For carnitine in the pet food it is seen that there is a significant difference in the measured results between days. Considering that a new calibration curve is Precision:

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made for every batch and that relative difference (daily average vs. overall aver- age) is low this is seen as being acceptable. For Carnitine the RSD% will be set to 5% for carnitine.

Trueness:

The methods trueness has been evaluated by analysing reference material. Reference materials: Reference materials with values on carnitine exist or are at the moment under testing for certification. We have analysed the certified reference material SRM 1849A (Infant/adult nutri- tional formula), SRM 1546 (Meat Homogenate) and SRM 1845A (Whole Egg

powder, under certification). Sample: NIST SRM 1849A Matrix: Infant/adult nutritional formula. Species Result (m) carnitine 140 ± 2 mg/kg 4 136 ± 14 mg/kg

n Reference value (CRM) k Comment

2 free The certified values have been evaluated according to ERM application Note 1. 1 Example of the calculation: The certified content of free carnitine is (136 ± 14) mg/kg, k = 2,00. The SRM 1849a was analysed 4 times. (140 ± 2) mg/kg.

If Δ m ≤ U Δ then no significant difference between the measurement result and the certified value has been demonstrated.

1 http://irmm.jrc.ec.europa.eu/html/reference_materials_catalogue/user_support/index.htm.

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4 ≤ 8 ,2 Therefor no difference between the results has been demonstrated on a 95% confidence level. To compare our results with data on 1849A and 1845A published at the 2 nd. Vita- min conference in Copenhagen 2 the data has been evaluated with at t-test (un- equal sample size, equal variance).

where

Using the zero hypotheses that no difference between the two data sets is ob- served. Sample: NIST SRM 1849A Matrix: Infant/adult nutritional formula. Species Result n Reference value n Comment carnitine 158 ± 4 mg/kg 8 149 ± 1 mg/kg 20 Total carnitine

At 26 degrees of freedom the t value of 2,0555 is critical for α=0,05

9 ≥ 2 ,1 The Zero hypotheses are rejected on a 95% confidence level. There is a signifi- cant difference between the results. The explanation is that alkaline hydrolysis is more efficient in hydrolysing the car-

2 Data presented at the 2 nd. International vitamin conference in Copenhagen 2012.

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nitine esters to form free carnitine than an acidic hydrolysis thereby giving higher results this is seen as ok because we desire to measure the total content of car- nitine. Sample: NIST SRM 1845A Measuring range: The measuring range in this validation covers the interval: LOQ – 1000 ng/g. Samples containing concentration higher than the highest standard will be reana- lysed with a more fitting sample size and/or dilution leading to a concentration in- side the standard range. LOD has been determined by treating a standard at low level (9 ng/g) as a sam- ple, the reason this approach has been used is that it has been impossible to find a natural sample containing carnitine at a level near the expected limit of detec- tion. The sample was analysed as a 10 replicates measurement on the same day, the standard deviation (S r ) has been calculated from these results. See appendix 3. LOD is calculated as 3 x S r and LOQ as (10 x S r ). The limit of detection and quantification for carnitine is calculated to: Limit of detection / quantification:

LOD: 0,9 ng/g LOQ: 3,0 ng/g

LOQ in samples as mg/kg is calculated from an aliquot of 20 g sample (maxi- mum) extracted into 110 g extraction solution. 10 g extraction solution is diluted to 250 g, (this sums up to a total dilution factor of 137,5). Carnitine in samples: LOQ: 0,4 mg/kg

Robustness:

The robustness of the method has been evaluated on the following critical pa- rameters; extraction time and saponification time. Extraction time: The robustness test has been performed on a milk powder and a pet food con-

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taining both carnitine. The sample is weighed into the extraction bottle, extraction solvents are added, the bottle is closed and placed on the shaking table at 37°C. At the time t. the sample is removed from the shaking table, centrifuged and placed on cold stor- age until saponification. t / hour 1 2 3 4 5 22 Carnitine:

Ekstraktion 37°C

100 150 200 250 300 350 400 Carnitine (mg/kg)

Pet food

Milk powder

0 50

Time (hour)

From the data (see appendix 4) it is seen that an extraction time of 20 hours is sufficient to extract all the carnitine from the sample. Saponification time: The robustness test has been performed on raw egg, pet food, milk powder (in- fant formula), cornflakes and fish feed containing both carnitine. An aliquot of the extraction solution is transferred to the saponification bottle, po- tassium hydroxide and water is added and the mixture is saponifyed at a 100°C. At the time t. the sample is removed from the oven and neutralized with hydro- chloric acid. 30 60 90 120 150 180 240 300 360 From the data (see appendix 4) it is seen that a saponification time of 240 min is sufficient to convert all bound carnitine to the free forms for all the tested ma- trixes. Carnitine: t / min 0 (free)

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Saponification 100°C

100 150 200 250 300 350 400 Carnitine (mg/kg)

Raw egg

Pet food

Infant formula

0 50

Cornflakes

Fishfeed

Time (min)

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Summary and conclusions:

The validation of the method has shown satisfactory specificity for the tested ma- trices. The calibration curves is linear for carnitine and they both going through the zero point (0;0). The precision experiments showed an acceptable RSD%, which was set to 5 %. The comparison between ‘this method’ and the values on the certified reference materials showed no significant differences. It is therefore concluded that ‘this method’ provides accurate results. Finally, ‘this method’ showed good robustness against the tested parameters. In general, we believe that the validation has been satisfactory.

Eurofins Steins Laboratory A/S Vitamin Competence Centre

28. of June 2013

6600 Vejen, Denmark

Lars T. Tanderup M.Sc. Chemistry

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Appendix 1: Linearity

Carnitine:

Actual Concentration (ng/g)

IS Actual Concentration (ng/g)

Area (counts)

IS Area (counts)

Concentration ratio

Area ratio

std 1 std 2 std 3 std 4 std 5 std 6 std 7 std 8 std 9 std 1 std 2 std 3 std 4 std 5 std 6 std 7 std 8 std 9

4,6129 9,0881 38,1799 74,0862 146,2597 256,4854 550,7591 1000,0994 1550,7960 4,6129 9,0881 38,1799 74,0862 146,2597 256,4854 550,7591 1000,0994 1550,7960

217,8116 225,4382 209,5343 221,8754 223,4094 218,8944 222,5433 221,0921 221,9617 217,8116 225,4382 209,5343 221,8754 223,4094 218,8944 222,5433 221,0921 221,9617

3329,5 7139,4

160220,7 181885,2

0,021178395 0,020780686 0,040313044 0,039252491 0,182213127 0,175655523 0,333909032 0,326066704 1,171731209 1,13343267 2,474840177 2,309526344 4,523451539 4,337842857 6,986772943 6,674058219 0,021178395 0,022730696 0,040313044 0,038167521 0,182213127 0,178401211 0,333909032 0,324567286 1,171731209 1,144839091 2,474840177 2,367995416 4,523451539 4,433062372 6,986772943 6,715054693 0,6546712 0,661242287 0,6546712 0,633634036

29263,0 166593,2 55518,4 170267,0 112370,8 169939,0 183940,7 162286,4 363396,5 157346,8 611060,3 140867,3 807552,3 120998,7 30022,4 168285,8 55438,6 170807,8 111210,2 175511,7 188626,3 164762,3 354499,4 149704,4 612058,0 138066,6 892823,7 132958,5 3769,3 6802,4 165826,3 178225,4

SUMMARY OUTPUT

Regression Statistics

Multiple R R Square

0,999913128

0,999826263 Adjusted R Square 0,999815404 Standard Error 0,030825052 Observations 18

ANAVA

df Significance F Regression 1 87,49023889 87,49023889 92077,17128 1,63032E-31 Residual 16 0,015202941 0,000950184 Total 17 87,50544183 SS MS F

Coefficients

Std. Error

t Stat

P-value

Lower 95%

Upper 95%

Intercept 0,00455914 0,009271008 0,491763144 0,629565074 -0,01509451 0,024212798 Concentration ratio 0,959609823 0,003162414 303,4422042 1,63032E-31 0,95290580 0,966313841

RESIDUAL OUTPUT Observation Predicted Area

Residuals

1 2 3 4 5 6 7 8

0,024882135 -0,004101449 0,043243933 -0,003991442 0,179412646 -0,003757124 0,324981527 0,001085177 0,632788054 0,028454233 1,128963918 0,004468752 2,379440084 -0,069913739 4,345307669 -0,007464813

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9

6,709135085 -0,035076867 0,024882135 -0,002151439 0,043243933 -0,005076411 0,179412646 -0,001011435 0,324981527 -0,000414241 0,632788054 0,000845982 1,128963918 0,015875173 2,379440084 -0,011444668 4,345307669 0,087754703 6,709135085 0,005919607

10 11 12 13 14 15 16 17 18

Calibration curve Carnitine

0 2 4 6 8

Area ratio

Area ratio

Linear (Area ratio)

0

2

4

6

8

Concentration ratio

Residualplot Carnitine

0.1

0.05

0

0

1

2

3

4

5

6

7

8

-0.05 Residualer

-0.1

Concentration ratio

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Appendix 2: Precision

Anava: Single Factor

Carnitine (mg/kg) Matrix: Milk powder

SUMMARY

Replicate 1 Replicate 2

Groups

Count

Sum

Average

Variance

day 1 day 2 day 3 day 4 day 5 day 6 day 7 day 8 day 9

96,110 99,537 97,998 98,114 98,540 100,585 95,046 98,207 90,559 95,917 99,272 93,529 92,832 91,049 92,619 92,152

95,648 95,202 97,719 93,736 98,514 93,959 95,060 94,833 96,481 89,388 99,268 93,463 92,560 91,474 95,317 95,635

day 1 day 2 day 3 day 4 day 5 day 6 day 7 day 8 day 9 day 10 day 11 day 12 day 13 day 14 day 15 day 16

2 191,758

95,879

0,106722

2 194,739 97,3695 9,3961125 2 195,717 97,8585 0,0389205

2 191,85 2 197,054 2 194,544 2 190,106 2 193,04 2 187,04 2 198,54 2 186,992 2 185,392

95,925 98,527

9,583442 0,000338

97,272 21,951938

95,053

9,8E-05 5,691938

96,52

93,52 17,535042

day 10 day 11 day 12 day 13 day 14 day 15 day 16

2 185,305 92,6525 21,3139205

99,27

8E-06

93,496 92,696

0,002178 0,036992

2 182,523 91,2615 0,0903125

2 187,936

93,968

3,639602

2 187,787 93,8935 6,0656445

Average of all days

95,32 mg/kg

ANAVA Source of Variation Between Groups

SS

df

MS

F

P-value

F crit

166,8678192 15 11,12452128 1,86470778 0,113837078 2,352222763

Within Groups

95,4532085 16 5,965825531

Total

262,3210277 31

Anava: Single Factor

Carnitine (mg/kg) Matrix: pet food

SUMMARY

Replicate 1 Replicate 2

Groups

Count

Sum

Average

Variance

day 1 day 2 day 3 day 4 day 5 day 6 day 7 day 8 day 9

357,418 346,842 342,908 348,298 353,781 318,183 365,650 363,560 366,707 336,970 342,770 348,137 343,212 330,134

352,315 347,401 354,980 351,989 360,330 336,589 368,775 361,314 349,201 341,202 349,700 335,804 344,259 343,909

day 1 day 2 day 3 day 4 day 5 day 6 day 7 day 8 day 9 day 10 day 11 day 12 day 13 day 14

2 709,733 354,8665 13,0203045 2 694,243 347,1215 0,1562405 2 697,888 348,944 72,866592 2 700,287 350,1435 6,8117405 2 714,111 357,0555 21,4447005 2 654,772 327,386 169,390418 2 734,425 367,2125 4,8828125

2 724,874 362,437

2,522258

2 715,908 357,954 153,230018

day 10 day 11 day 12 day 13 day 14

2 678,172 339,086 2 692,47 346,235

8,954912 24,01245

2 683,941 341,9705 76,0514445 2 687,471 343,7355 0,5481045 2 674,043 337,0215 94,8753125

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day 15 day 16

338,625 332,365

342,389 340,596

day 15 day 16

2 681,014 340,507

7,083848

2 672,961 336,4805 33,8746805

Average of all days

347,38 mg/kg

ANAVA Source of Variation Between Groups

SS

df

MS

F

P-value

F crit

3354,811135 15 223,6540757 5,188242953 0,001098565 2,352222763

Within Groups

689,7258365 16 43,10786478

Total

4044,536971 31

Appendix 3: Limit of detection / quantification

Carnitine:

Replicate

9,414 ng/g 9,379 ng/g 9,145 ng/g 9,247 ng/g 9,418 ng/g 9,896 ng/g 9,409 ng/g 9,432 ng/g 9,810 ng/g 10,080 ng/g

Mean 9,523 ng/g Std 0,301 ng/g

LOD 0,9 ng/g LOQ 3,0 ng/g

In samples LOD 124 ng/g = 0,1 µg/g LOQ 413 ng/g = 0,4 µg/g

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Appendix 4: Robustness.

Ekstraktion of carnitine:

Time Pet food Milk powder (hour) Carnitine (mg/kg) Carnitine (mg/kg) 1 322 85 2 328 84 3 346 84 4 354 85 5 348 87 22 368 84

Saponification of carnitine:

Time Raw egg Fish feed (min) Carnitine (mg/kg) Carnitine (mg/kg) Carnitine (mg/kg) Carnitine (mg/kg) Carnitine (mg/kg) 0 11 361 91 6 43 30 27 335 86 6 48 60 12 343 84 7 51 90 14 356 86 6 49 120 12 355 86 6 51 150 48 340 85 6 51 180 13 338 86 6 52 240 22 351 83 5 52 300 12 350 86 6 51 360 13 350 89 5 50 Pet food Milk powder Cornflakes

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Eurofins Steins A/S Validation Report: Addendum SPIFAN kit Carnitine quantitated by liquid chromatography and isotope dilu- tion mass spectroscopy.

Lars T. Tanderup Chemist M.Sc.

Eurofins Steins Laboratory A/S Vitamin Competence Centre 6600 Vejen, Denmark 2014-10-01

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Table of Contents:

Abbreviations and synonyms: .....................................................................................................................................1 Quality statement:........................................................................................................................................................1 Introduction:.................................................................................................................................................................1 Statistics: .....................................................................................................................................................................1 Matrices: ......................................................................................................................................................................1 Precision:.....................................................................................................................................................................2 Appendix 1:..................................................................................................................................................................4

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Abbreviations and synonyms:

Abbreviations: NMKL.................Nordisk Metodikk Komité for Næringsmidler ISO ....................International Organization for Standardization SOP...................Standard Operating Procedure ANAVA ..............Analysis of Variance n ........................Number of replicates RSD%................Relative Standard Deviation in Percent CI .......................Confidence intervals S r .......................Repeatability (standard deviation within days) S R ......................Reproducibility (standard deviation between days) Synonyms: t-test .................. Student’s t -test This validation report is conducted in accordance to our internal instruction 211.01 “Validering af analysemetoder i Vitamin center (in English - Validation of analytical methods in the Vitamin Centre” This instruction defines the experimental design used in the current validation, which is based on NMKL Procedure No. 4: Validation of chemical analytical methods (2009, 3 rd edition). The Eurofins Steins Laboratory in Vejen is accredited according to ISO 17025. Quality statement:

Introduction:

The carnitine method has been employed on the SPIFAN test kit to investigate the methods capabilities on this set of samples.

Statistics:

In the current validation report, a p-value < 0.05 is considered statistically signifi- cant. Consequently, confidence intervals (CI) are calculated at a confidence level at 95 %. Paired t-testing is performed on normal distributed raw data. Data which is not within the normal distribution is clearly marked within the text.

Matrices:

The SPIFAN test kit consists of 17 samples providing a representative collection

Page 1 of 23

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of Infant and nutritional formulas, both as powder and ready to feed. The samples have been tested for both total and free carnitine.

Precision:

The data consists of 3 days of double replicate measurements. The precision have been evaluated with a variance analysis model. (ANAVA single factor (Excel 2010), for data see appendix 1.)

Total carnitine: Sample name Child Formula Powder Infant Element Powder

Lot#

Result (mg/kg) RSD% n

00411RF00 00402RF00 00415RF00 00407RF00

2,4 0,2 0,4 0,5

9,1 6 74 6 17 6 10 6 2,0 6 2,3 6 30 6 3,2 6 1,8 6 17 6 1,9 5 2,0 6 1,8 6 3,5 6 1,5 6 0,8 6 1,2 6 9,1 6 39 6 10 6 19 6 2,1 6 2,7 6 12 6 2,7 6 2,3 6 11 6 0,7 5 1,3 6 1,7 6 1,7 6 1,4 6 2,5 6 2,2 6 10,3 6

Adult Nut. RFT High Protein Adult Nut. RFT High Fat

Infant Formula RFT, Milk Based Spifan Blank Milk Form

26,9

SRM 1849a

CLC10-b

151,8

Adult Nut. Protein Based

11750017V3 1172572116 117257651Z 00394RF00 000412RF00 000403RF00 D04HTCVV

0,9

Infant Formula, Part. Hyd. Milk Infant Formula, Part. Hyd. Soy

105,3

93,3

Adult Nut. Low Fat Child Formula Infant Elemental

3,3

490,5 137,4 159,1

Infant Formula, Milk Based Infant Formula, Soy Based

E29JVLV

82,8 27,9

Infant Formula RFT, Milk Based Spifan Control Milk Form

Adult Nut. RFT High Protein Adult Nut. RFT High Fat

000414RF00 00406RF00 00411RF00

156,2 221,7

Child Formula Powder

2,4

Free carnitine: Sample name Child Formula Powder Infant Element Powder

Lot#

Result (mg/kg) RSD% n

00411RF00 00402RF00 00415RF00 00407RF00

1,5 0,4 0,4 0,5

Adult Nut. RFT High Protein Adult Nut. RFT High Fat

Infant Formula RFT, Milk Based Spifan Blank Milk Form

25,1

SRM 1849a

CLC10-b

130,8

Adult Nut. Protein Based

11750017V3 1172572116 117257651Z 00394RF00 000412RF00 000403RF00 D04HTCVV

0,7

Infant Formula, Part. Hyd. Milk Infant Formula, Part. Hyd. Soy

81,9 91,8

Adult Nut. Low Fat Child Formula Infant Elemental

2,1

484,5 135,7 138,5

Infant Formula, Milk Based Infant Formula, Soy Based

E29JVLV

81,6 26,0

Infant Formula RFT, Milk Based Spifan Control Milk Form

Adult Nut. RFT High Protein Adult Nut. RFT High Fat

000414RF00 00406RF00

152,6 216,7

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Child Formula Powder

00411RF00

1,5

10,3 6

Eurofins Steins Laboratory A/S Vitamin Competence Centre

1. of October 2014

6600 Vejen, Denmark

Lars T. Tanderup M.Sc. Chemistry

Carn-06 (Validation Report-Addendum) FOR ERP USE ONLY DO NOT DISTRIBUTE

Appendix 1:

Total Carnitne:

Sample name

Lot#

Day 1 (mg/kg)

Day 2 (mg/kg)

Day 3 (mg/kg)

Child Formula Powder Child Formula Powder Infant Element Powder Infant Element Powder Adult Nut. RFT High Pro- tein Adult Nut. RFT High Pro- tein Adult Nut. RFT High Fat Adult Nut. RFT High Fat Infant Formula RFT, Milk Based Infant Formula RFT, Milk Based Adult Nut. Protein Based Adult Nut. Protein Based Infant Formula, Part. Hyd. Milk Infant Formula, Part. Hyd. Milk Infant Formula, Part. Hyd. Soy Infant Formula, Part. Hyd. Soy SRM 1849a SRM 1849a

00411RF00 00411RF00 00402RF00 00402RF00 00415RF00 00415RF00 00407RF00 00407RF00 Spifan Blank Milk Form Spifan Blank Milk Form

Rep. 1 Rep. 2 Rep. 1 Rep. 2 Rep. 1 Rep. 2 Rep. 1 Rep. 2 Rep. 1 Rep. 2 Rep. 1 Rep. 2 Rep. 1 Rep. 2 Rep. 1 Rep. 2 Rep. 1 Rep. 2 Rep. 1 Rep. 2 Rep. 1 Rep. 2 Rep. 1 Rep. 2 Rep. 1 Rep. 2 Rep. 1 Rep. 2 Rep. 1

2,473703509 2,355708642 2,509208389 2,689167018 2,570025549 2,043636402 0,104853161 0,271640946 0,397308668 0 0,327039839 0,174439427 0,374729502 0,423287923 0,319515997 0,450900581 0,274724682 0,371810272 0,524927193 0,425687194 0,573019819 0,49715477 0,503670598 0,530655394 26,78003995 27,18015772 26,36241845 27,05015009 26,52216338 27,94984392 157,2860134 152,0292092 151,9379274 153,0955396 148,7525652 147,7333929 1,238787861 0,797090813 1,196324638 0,618322033 0,693928605 1,186500537

CLC10-b CLC10-b

11750017V3 11750017V3 1172572116

101,7408897

105,950458

108,560353

1172572116

102,7245457 109,4827952 103,1222465

117257651Z

92,0471692 94,53785472

94,638898

117257651Z 00394RF00 00394RF00 000412RF00 000412RF00 000403RF00 000403RF00

92,14563536 91,33048028 94,85876736 2,869795754 3,254945707 3,383791845 2,780390073 4,293968674 3,383221091 502,5701183 491,1207734 478,2288395 133,5507137 138,1377884 138,5474767 135,737155 140,7826542 137,7373237 155,1631811 159,4015029 163,9026204 159,3621359 157,6750995 159,0079721 83,68006675 80,86826851 88,22903551 80,78305012 83,21631811 79,85014519 495,3545363 497,5476247 lost in lab

Adult Nut. Low Fat Adult Nut. Low Fat Child Formula Child Formula Infant Elemental Infant Elemental

Infant Formula, Milk Based D04HTCVV Infant Formula, Milk Based D04HTCVV Infant Formula, Soy Based E29JVLV Infant Formula, Soy Based E29JVLV

Infant Formula RFT, Milk Based Infant Formula RFT, Milk Based Adult Nut. RFT High Pro- tein Adult Nut. RFT High Pro- tein Adult Nut. RFT High Fat Adult Nut. RFT High Fat

Spifan Control Milk Form Spifan Control Milk Form 000414RF00 000414RF00 00406RF00 00406RF00

27,7066557 27,89329797

28,116869

Rep. 2

28,11958283 27,11989578 28,18837915

Rep. 1

154,3249916 155,8793288 157,3248917

Rep. 2 Rep. 1 Rep. 2

157,2425161 155,4211802 156,9057613 220,6765481 220,4129711 221,6249524 218,1061917 223,8276604 225,4660592

Sample name: Child Formula Powder Eurofins Sample nr.: 487-2014-04020186

Rep. 1 (mg/kg) Rep. 2 (mg/kg) 2,473703509 2,689167018 2,355708642 2,570025549

Average of all days

Day 1 Day 2

2,44 mg/kg

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Day 3

2,509208389 2,043636402

Anava: Single faktor Summary Groups

Count

Sum

Average

Variance

Day 1 Day 2 Day 3

2 5,162870528 2,581435264 0,023212262 2 4,925734191 2,462867096 0,022965868 2 4,552844791 2,276422395 0,108378638

ANAVA Source of Variation

SS

df

MS

F

P-value

F crit

Between Groups Within Groups

0,094568591 0,154556768 0,249125359 -0,002117314 0,049401609

2 0,047284296 0,917804431 0,488658 9,552094

3 0,051518923

Total Sl^2 SR^2

5

Reproducibility (SR) Repeatability (Sr) Sample name: Infant Element Powder Eurofins Sample nr.: 487-2014-04020187

0,22 mg/kg 0,23 mg/kg

RSD%

9,1 % 9,3 %

Rep 1 (mg/kg) Rep 2 (mg/kg)

Average of all days

Day 1 Day 2 Day 3

0,104853161

0

0,21 mg/kg

0,271640946 0,327039839 0,397308668 0,174439427

Anava: Single faktor Summary Groups

Count

Sum

Average

Variance

Day 1 Day 2 Day 3

2 0,104853161 0,05242658 0,005497093 2 0,598680785 0,299340393 0,001534519 2 0,571748095 0,285874047 0,024835349

ANAVA Source of Variation

SS

df

MS

F

P-value

F crit

Between Groups Within Groups

0,077096995 0,031866961 0,108963956 0,013963089 0,024585409

2 0,038548498 3,629009177 0,158156 9,552094

3 0,01062232

Total Sl^2 SR^2

5

Reproducibility (SR) Repeatability (Sr)

0,16 mg/kg 0,10 mg/kg

RSD%

73,8 % 48,5 %

Sample name Adult Nut. RFT High Protein Sample nr. 487-2014-04020188

Rep 1 (mg/kg) Rep 2 (mg/kg) 0,374729502 0,450900581 0,423287923 0,274724682 0,319515997 0,371810272

Average of all days

Day 1 Day 2 Day 3

0,37 mg/kg

Anava: Single faktor Summary

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Groups

Count

Sum

Average

Variance

Day 1 Day 2 Day 3

2 0,825630083 0,412815042 0,002901017 2 0,698012605 0,349006302 0,011035518 2 0,691326269 0,345663134 0,001367346

ANAVA Source of Variation

SS

df

MS

F

P-value

F crit

Between Groups Within Groups

0,005728074 0,015303881 0,021031954 -0,001118628 0,003982665

2 0,002864037 0,561433461 0,620702 9,552094

3 0,005101294

Total Sl^2 SR^2

5

Reproducibility (SR) Repeatability (Sr)

0,06 mg/kg 0,07 mg/kg

RSD%

17,1 % 19,3 %

Sample name Adult Nut. RFT High Fat Sample nr. 487-2014-04020189

Rep 1 (mg/kg) Rep 2 (mg/kg)

Average of all days

Day 1 Day 2 Day 3

0,524927193

0,49715477

0,51 mg/kg

0,425687194 0,503670598 0,573019819 0,530655394

Anava: Single faktor Summary Groups

Count

Sum

Average

Variance

Day 1 Day 2 Day 3

2 1,022081963 0,511040981 0,000385654 2 0,929357791 0,464678896 0,003040706 2 1,103675213 0,551837606 0,000897372

ANAVA Source of Variation

SS

df

MS

F

P-value

F crit

Between Groups Within Groups

0,007606966 0,004323732 0,011930697 0,001181119 0,002622363

2 0,003803483 2,639027882 0,218167 9,552094

3 0,001441244

Total Sl^2 SR^2

5

Reproducibility (SR) Repeatability (Sr)

0,05 mg/kg 0,04 mg/kg

RSD%

10,1 % 7,5 %

Sample name Infant Formula RFT, Milk Based Sample nr. 487-2014-04020190

Rep 1 (mg/kg) Rep 2 (mg/kg) 26,78003995 27,05015009 27,18015772 26,52216338 26,36241845 27,94984392

Average of all days

Day 1 Day 2 Day 3

26,97 mg/kg

Anava: Single faktor Summary Groups

Count

Sum

Average

Variance

Day 1 Day 2 Day 3

2 53,83019003 26,91509502 0,036479743 2 53,70232111 26,85116055 0,216478276 2 54,31226236 27,15613118 1,25995981

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ANAVA Source of Variation

SS

df

MS

F

P-value

F crit

Between Groups Within Groups

0,103462088 1,51291783 1,616379917 -0,22628745 0,278018494

2 0,051731044 0,102578692 0,90554 9,552094

3 0,504305943

Total Sl^2 SR^2

5

Reproducibility (SR) Repeatability (Sr) Sample name SRM 1849a Sample nr. 487-2014-04020191

0,53 mg/kg 0,71 mg/kg

RSD%

2,0 % 2,6 %

Rep 1 (mg/kg) Rep 2 (mg/kg) 157,2860134 153,0955396 152,0292092 148,7525652 151,9379274 147,7333929

Average of all days

Day 1 Day 2 Day 3

151,81 mg/kg

Anava: Single faktor Summary Groups

Count

Sum

Average

Variance

Day 1 Day 2 Day 3

2 310,381553 155,1907765 8,780035677 2 300,7817745 150,3908872 5,368198018 2 299,6713202 149,8356601 8,83905511

ANAVA Source of Variation

SS

df

MS

F

P-value

F crit

Between Groups Within Groups

34,68299025 22,98728881 57,67027906 4,839532762 12,50196236

2 17,34149513 2,26318492 0,251654 9,552094

3 7,662429602

Total Sl^2 SR^2

5

Reproducibility (SR) Repeatability (Sr)

3,54 mg/kg 2,77 mg/kg

RSD%

2,3 % 1,8 %

Sample name Adult Nut. Protein Based Sample nr. 487-2014-04020192

Rep 1 (mg/kg) Rep 2 (mg/kg) 1,238787861 0,618322033 0,797090813 0,693928605 1,196324638 1,186500537

Average of all days

Day 1 Day 2 Day 3

0,96 mg/kg

Anava: Single faktor Summary Groups

Count

Sum

Average

Variance

Day 1 Day 2 Day 3

2 1,857109894 0,928554947 0,192488922 2 1,491019418 0,745509709 0,005321221 2 2,382825174 1,191412587 4,82565E-05

ANAVA Source of Variation

SS

df

MS

F

P-value

F crit

Between Groups Within Groups

0,200952717 0,197858399

2 0,100476358 1,523458576 0,349447 9,552094

3

0,0659528