AbsoluteIDQ™ Kit Analytical Specifications

Contents General Remarks .................................................................................................. 1

Procedural Notes................................................................................................. 1 Limitations of The AbsoluteIDQ™ Kit ................................................................. 1 Calculation of Results.......................................................................................... 2 Quality Control..................................................................................................... 3

Analytical Performance Characteristics ............................................................. 3 Overview ............................................................................................................. 3 Specificity .......................................................................................................... 11 Linearity, Precision and Accuracy ..................................................................... 13 Functional Sensitivity......................................................................................... 13 Reproducibility ................................................................................................... 14 Stability.............................................................................................................. 14

AbsoluteIDQ™ Kit

The information in this document is subject to change without notice and should not be construed as a commitment by Biocrates Life Sciences AG to assume responsibility for any errors that may appear. These product specifications are believed to be accurate for use in working with the AbsoluteIDQ Kit™. In no event shall Biocrates Life Sciences AG be liable for incidental or consequential damages in connection with or arising from the use of these analytical specifications. The AbsoluteIDQ Kit™ is for research use only. It is not for use in diagnostic procedures. For brevity, AbsoluteIDQ Kit may be used in place of the full trademark name AbsoluteIDQ™ Kit in descriptions and tables in this book.

PN: 4004-P150-AS-1

Analytical Specifications Page 1

General Remarks The analytical principle of the AbsoluteIDQ Kit is based on a complex combination of experimental steps, namely the extraction of analytes, derivatization and mass spectrometric detection via ESI-MS/MS (FIA). The complexity of the process gives rise to:

i) the necessity to follow the user manual in detail, and ii) method-dependant limitations of the analysis.

Procedural Notes

1. To successfully use the AbsoluteIDQ Kit for your metabolomics research, make sure that you thoroughly understand the components of the Kit.

2. Be aware that any deviation from the assay procedure may affect the results. In particular, it has to be taken into account that this kit product is specified for use only with human plasma samples.

3. So as not to waste costly sample material, make sure that you are familiar with the MetIQ software and that your analytical system meets the requirements before running the assay in the lab. Please consult the BIOCRATES support team for special Starter Kit options to help you to get started, if needed.

4. We do not recommend re-using standard solutions or quality control material. Also, you should use only original kit reagents and contents for your assay.

5. Do not hesitate to contact our customer support in case you had questions about the assay, handling or sample preparation (support@biocrates.com).

Limitations of the AbsoluteIDQ™ Kit Please be aware that the Kit represents a device for metabolomics research. Hence the analytical focus of the Kit lies on measuring a very broad spectrum of metabolites in an easy, reliable and robust way which is, moreover, capable of being automated. The flow injection ESI-MS/MS detection and the extraction method used in this Kit limit the range of analytes that can be quantified. With this respect, the Kit was developed with the goal to find a reasonable compromise between analytical speed, robustness and quantitative measures. As a consequence, not all members of a metabolite class that are biochemically or medically important can be detected in an analytically valid manner by the Kit. However, to deliver broadest information possible, it was decided to incorporate a number of

Page 2 Analytical Specifications

AbsoluteIDQ™ Kit

analytes that are not meeting general validation criteria according to the FDA Guidance for Industry - Bioanalytical Method Validation. Nevertheless, these analytes exhibited robust values with good variance criteria and are especially labelled in the following lists of analytical specifications. Furthermore, in terms of quantification a subset of analytes is called “semi-quantitative” due to the fact that specific standards were not commercially available. In addition, some values display sum quantities of isobaric analytes. The MetIQ software reflects this by exhibiting a list of possible short names wheras the name of the most reasonable biological compound is printed in bold fonts. From BIOCRATES experience during development and field testing, these general conditions are known to cause anomalous results:

• Contamination of the instrumentation: autosampler, capillaries, tubing. • Imprecise pipetting of sample or control material. • Inhomogenous sample material, (like clotted plasma). • Uncontrolled storage of sample material before running the assay.

Calculation of Results

The MetIQ software is an integrated part of the AbsoluteIDQ Kit. A special algorithm for the calculation of analyte concentrations out of the MS spectra makes use of the Analyst software and is incorporated in the MetIQ module MetConc. The values are calculated by comparing the measured analytes in a defined section of the XIC to those of the internal standards which are provided by the kit plate. To assess the quality of the measurement a couple of tests are built into the software. In the case of non-valid analyses the MetVal as well as the MetStat module display the affected validation criteria. The following ranking for the different causes that may happen is pre-defined: Blank Out of Range > STD Out of Range > QC Out of Range > ISTD Out of Range > Out of Quant. All data including the validation criteria are displayed in a result table in MetStat. To generate meaningful metabolomics insight profound statistical analysis is necessary. Therefore, the data need to be exported to suitable statistics software.

Analytical Specifications Page 3

Quality Control

There are three different quality controls to be positioned as indicated in the User Manual. The three different controls represent human plasma samples (anticoagulant: EDTA) with three different concentration levels of spiked compounds (low, medium, high) – one compound per metabolite class except for hexoses. Results can be displayed in the MetVal module of MetIQ and are visualized in the result tables of MetStat module in MetIQ.

Analytical Performance Characteristics

Overview

This version of the AbsoluteIDQ Kit p150 measures the analytes as summarized in Tables 1.1 through 1.5. The concentration values for all analytes labelled with one asterisk in the Quality Criterion column produce semi-quantitative data due to the fact that standards were not available. In most cases, this means that accuracies could not be determined over the full quantification range though the data were highly reproducible. Analytes labelled with two asterisks in the Quality Criterion column failed to meet some validation criteria during analytical validation (for details see below). This means at least one criterion was out of the predefined range; however, all concentrations exhibit good reproducibility and may therefore be used for relative metabolomics approaches. Tables of all analytes measured by the AbsoluteIDQ Kit p150. Table 1.1 Acylcarnitines Table 1.2 Amino acids Table 1.3 Sugars Table 1.4 Glycerophospholipids Table 1.5 Sphingolipids

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AbsoluteIDQ™ Kit

Table 1.1 Acylcarnitines

Analyte Quantification Range MetIQ Short Name Biochemical Name

Quality Criterion Lower Limit (µM) Upper Limit (µM)

1 C0 DL-Carnitine 1.520 60.80 2 C2 Acetyl-L-carnitine 0.380 15.20 3 C3 Propionyl-L-carnitine 0.076 1.52 4 C3:1 Propenoyl-L-carnitine * 0.610 1000 5 C3-OH Hydroxypropionyl-L-carnitine * 0.610 1000 6 C4 Butyryl-L-carnitine 0.076 1.52 7 C4:1 Butenyl-L-carnitine * 0.010 1000 8 C4-OH Hydroxybutyryl-L-carnitine * 0,050 1000 9 C5 Valeryl-L-carnitine 0.076 1.52 10 C5:1 Tiglyl-L-carnitine * 0.100 1000 11 C5:1-DC Glutaconyl-L-carnitine * 0.070 1000

12 C5-DC (C6-OH) Glutaryl-L-carnitine (Hydroxyhexanoyl-L-carnitine) * 0.070 1000

13 C5-M-DC Methylglutaryl-L-carnitine * 2.930 1000

14 C5-OH (C3-DC-M) Hydroxyvaleryl-L-carnitine (Methylmalonyl-L-carnitine) * 1.680 1000

15 C6 (C4:1-DC) Hexanoyl-L-carnitine (Fumaryl-L-carnitine)

0.152 3.04

16 C6:1 Hexenoyl-L-carnitine * 0.100 1000 17 C7-DC Pimelyl-L-carnitine * 0.200 1000 18 C8 Octanoyl-L-carnitine 0.152 3.04 19 C8:1 Octenoyl-L-carnitine * 0.110 1000 20 C9 Nonayl-L-carnitine * 0.290 1000 21 C10 Decanoyl-L-carnitine 0.152 3.04 22 C10:1 Decenoyl-L-carnitine * 0.440 1000 23 C10:2 Decadienyl-L-carnitine * 0.850 1000 24 C12 Dodecanoyl-L-carnitine 0.152 3.04 25 C12:1 Dodecenoyl-L-carnitine * 0.680 1000

Analytical Specifications Page 5

Analyte Quantification Range MetIQ Short Name Biochemical Name

Quality Criterion Lower Limit (µM) Upper Limit (µM)

26 C14 Tetradecanoyl-L-carnitine 3.040 6.08 27 C14:1 Tetradecenoyl-L-carnitine * 0.070 1000

28 C14:1-OH Hydroxytetradecenoyl-L-carnitine * 0.190 1000

29 C14:2 Tetradecadienyl-L-carnitine * 0.090 1000

30 C14:2-OH Hydroxytetradecadienyl-L-carnitine * 0.040 1000

31 C16 Hexadecanoyl-L-carnitine 0.304 6.08 32 C16:1 Hexadecenoyl-L-carnitine * 0.110 1000

33 C16:1-OH Hydroxyhexadecenoyl-L-carnitine * 0.090 1000

34 C16:2 Hexadecadienyl-L-carnitine * 0.040 1000

35 C16:2-OH Hydroxyhexadecadienyl-L-carnitine * 0.100 1000

36 C16-OH Hydroxyhexadecanoyl-L-carnitine * 0.050 1000

37 C18 Octadecanoyl-L-carnitine 0.152 6.08 38 C18:1 Octadecenoyl-L-carnitine * 0.280 1000

39 C18:1-OH Hydroxyoctadecenoyl-L-carnitine * 0.070 1000

40 C18:2 Octadecadienyl-L-carnitine * 0.050 1000

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AbsoluteIDQ™ Kit

Table 1.2 Amino Acids

Analyte Quantification Range MetIQ Short Name Biochemical Name

Quality Criterion Lower Limit (µM) Upper Limit (µM)

41 Arg-PTC Arginine ** 5.0 200 42 Gln-PTC Glutamine ** 3.2 1000 43 Gly-PTC Glycine * 0.01 1000 44 His-PTC Histidine 5.0 200 45 Met-PTC Methionine 5.0 200 46 Orn-PTC Ornithine 5.0 150 47 Phe-PTC Phenylalanine 5.0 200 48 Pro-PTC Proline 5.0 200 49 Ser-PTC Serine 5.0 200 50 Thr-PTC Threonine * 0.1 500 51 Trp-PTC Tryptophan 50.0 200 52 Tyr-PTC Tyrosine * 5.0 200 53 Val-PTC Valine 5.0 200 54 xLeu-PTC xLeucine * 5.0 200

Table 1.3 Sugars

Analyte Quantification Range MetIQ Short Name Biochemical Name

Quality Criterion Lower Limit (µM) Upper Limit (µM)

55 H1 Hexose * 200 10000

Analytical Specifications Page 7

Table 1.4 Glycerophospholipids

Analyte Quantification Range MetIQ Short Name Biochemical Name

Quality Criterion Lower Limit (µM) Upper Limit (µM)

56 lysoPC a C6:0 lysoPhosphatidylcholine acyl C6:0 * 0.10 1000 57 lysoPC a C14:0 lysoPhosphatidylcholine acyl C14:0 * 145 1000 58 lysoPC a C16:0 lysoPhosphatidylcholine acyl C16:0 * 5.84 1000 59 lysoPC a C16:1 lysoPhosphatidylcholine acyl C16:1 * 0.62 1000 60 lysoPC a C17:0 lysoPhosphatidylcholine acyl C17:0 * 0.17 1000 61 lysoPC a C18:0 lysoPhosphatidylcholine acyl C18:0 * 2.25 1000 62 lysoPC a C18:1 lysoPhosphatidylcholine acyl C18:1 * 1.58 1000 63 lysoPC a C18:2 lysoPhosphatidylcholine acyl C18:2 * 2.43 1000 64 lysoPC a C20:3 lysoPhosphatidylcholine acyl C20:3 * 1.25 1000 65 lysoPC a C20:4 lysoPhosphatidylcholine acyl C20:4 * 1.25 1000 66 lysoPC a C24:0 lysoPhosphatidylcholine acyl C24:0 * 8.41 1000 67 lysoPC a C26:0 lysoPhosphatidylcholine acyl C26:0 * 2.09 1000 68 lysoPC a C26:1 lysoPhosphatidylcholine acyl C26:1 * 19.51 1000 69 lysoPC a C28:0 lysoPhosphatidylcholine acyl C28:0 * 4.90 1000 70 lysoPC a C28:1 lysoPhosphatidylcholine acyl C28:1 * 1.39 1000 71 PC aa C24:0 Phosphatidylcholine diacyl C24:0 * 0.33 1000 72 PC aa C26:0 Phosphatidylcholine diacyl C26:0 * 3.60 1000 73 PC aa C28:1 Phosphatidylcholine diacyl C28:1 * 0.28 1000 74 PC aa C30:0 Phosphatidylcholine diacyl C30:0 * 1.22 1000 75 PC aa C30:2 Phosphatidylcholine diacyl C30:2 * 1.44 1000 76 PC aa C32:0 Phosphatidylcholine diacyl C32:0 * 0.58 1000 77 PC aa C32:1 Phosphatidylcholine diacyl C32:1 * 1.33 1000 78 PC aa C32:2 Phosphatidylcholine diacyl C32:2 * 0.59 1000 79 PC aa C32:3 Phosphatidylcholine diacyl C32:3 * 0.09 1000 80 PC aa C34:1 Phosphatidylcholine diacyl C34:1 * 8.72 1000 81 PC aa C34:2 Phosphatidylcholine diacyl C34:2 * 12.84 1000 82 PC aa C34:3 Phosphatidylcholine diacyl C34:3 * 0.64 1000 83 PC aa C34:4 Phosphatidylcholine diacyl C34:4 * 0.08 1000 84 PC aa C36:0 Phosphatidylcholine diacyl C36:0 * 1.02 1000 85 PC aa C36:1 Phosphatidylcholine diacyl C36:1 * 2.67 1000

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AbsoluteIDQ™ Kit

Analyte Quantification Range MetIQ Short Name Biochemical Name

Quality Criterion Lower Limit (µM) Upper Limit (µM)

86 PC aa C36:2 Phosphatidylcholine diacyl C36:2 * 8.67 1000. 87 PC aa C36:3 Phosphatidylcholine diacyl C36:3 * 5.19 1000 88 PC aa C36:4 Phosphatidylcholine diacyl C36:4 * 6.68 1000 89 PC aa C36:5 Phosphatidylcholine diacyl C36:5 * 0.94 1000 90 PC aa C36:6 Phosphatidylcholine diacyl C36:6 * 0.13 1000 91 PC aa C38:0 Phosphatidylcholine diacyl C38:0 * 0.28 1000 92 PC aa C38:1 Phosphatidylcholine diacyl C38:1 * 0.38 1000 93 PC aa C38:3 Phosphatidylcholine diacyl C38:3 * 2.28 1000 94 PC aa C38:4 Phosphatidylcholine diacyl C38:4 * 3.56 1000 95 PC aa C38:5 Phosphatidylcholine diacyl C38:5 * 2.04 1000 96 PC aa C38:6 Phosphatidylcholine diacyl C38:6 * 2.45 1000 97 PC aa C40:1 Phosphatidylcholine diacyl C40:1 * 0.67 1000 98 PC aa C40:2 Phosphatidylcholine diacyl C40:2 * 0.11 1000 99 PC aa C40:3 Phosphatidylcholine diacyl C40:3 * 0.06 1000 100 PC aa C40:4 Phosphatidylcholine diacyl C40:4 * 0.19 1000 101 PC aa C40:5 Phosphatidylcholine diacyl C40:5 * 0.47 1000 102 PC aa C40:6 Phosphatidylcholine diacyl C40:6 * 2.34 1000 103 PC aa C42:0 Phosphatidylcholine diacyl C42:0 * 0.48 1000 104 PC aa C42:1 Phosphatidylcholine diacyl C42:1 * 0.06 1000 105 PC aa C42:2 Phosphatidylcholine diacyl C42:2 * 0.02 1000 106 PC aa C42:4 Phosphatidylcholine diacyl C42:4 * 0.08 1000 107 PC aa C42:5 Phosphatidylcholine diacyl C42:5 * 0.18 1000 108 PC aa C42:6 Phosphatidylcholine diacyl C42:6 * 0.90 1000 109 PC ae C30:0 Phosphatidylcholine acyl-alkyl C30:0 * 0.71 1000 110 PC ae C30:1 Phosphatidylcholine acyl-alkyl C30:1 * 0.12 1000 111 PC ae C30:2 Phosphatidylcholine acyl-alkyl C30:2 * 0.96 1000 112 PC ae C32:1 Phosphatidylcholine acyl-alkyl C32:1 * 0.12 1000 113 PC ae C32:2 Phosphatidylcholine acyl-alkyl C32:2 * 0.07 1000 114 PC ae C34:0 Phosphatidylcholine acyl-alkyl C34:0 * 0.35 1000 115 PC ae C34:1 Phosphatidylcholine acyl-alkyl C34:1 * 0.35 1000 116 PC ae C34:2 Phosphatidylcholine acyl-alkyl C34:2 * 0.40 1000 117 PC ae C34:3 Phosphatidylcholine acyl-alkyl C34:3 * 0.28 1000

Analytical Specifications Page 9

Analyte Quantification Range MetIQ Short Name Biochemical Name

Quality Criterion Lower Limit (µM) Upper Limit (µM)

118 PC ae C36:0 Phosphatidylcholine acyl-alkyl C36:0 * 0.63 1000 119 PC ae C36:1 Phosphatidylcholine acyl-alkyl C36:1 * 0.38 1000 120 PC ae C36:2 Phosphatidylcholine acyl-alkyl C36:2 * 0.55 1000 121 PC ae C36:3 Phosphatidylcholine acyl-alkyl C36:3 * 0.32 1000 122 PC ae C36:4 Phosphatidylcholine acyl-alkyl C36:4 * 0.49 1000 123 PC ae C36:5 Phosphatidylcholine acyl-alkyl C36:5 * 0.40 1000 124 PC ae C38:0 Phosphatidylcholine acyl-alkyl C38:0 * 0.17 1000 125 PC ae C38:1 Phosphatidylcholine acyl-alkyl C38:1 * 0.26 1000 126 PC ae C38:2 Phosphatidylcholine acyl-alkyl C38:2 * 0.48 1000 127 PC ae C38:3 Phosphatidylcholine acyl-alkyl C38:3 * 0.33 1000 128 PC ae C38:4 Phosphatidylcholine acyl-alkyl C38:4 * 0.45 1000 129 PC ae C38:5 Phosphatidylcholine acyl-alkyl C38:5 * 0.55 1000 130 PC ae C38:6 Phosphatidylcholine acyl-alkyl C38:6 * 0.30 1000 131 PC ae C40:0 Phosphatidylcholine acyl-alkyl C40:0 * 15.20 1000 132 PC ae C40:1 Phosphatidylcholine acyl-alkyl C40:1 * 0.17 1000 133 PC ae C40:2 Phosphatidylcholine acyl-alkyl C40:2 * 0.11 1000 134 PC ae C40:3 Phosphatidylcholine acyl-alkyl C40:3 * 0.11 1000 135 PC ae C40:4 Phosphatidylcholine acyl-alkyl C40:4 * 0.12 1000 136 PC ae C40:5 Phosphatidylcholine acyl-alkyl C40:5 * 0.15 1000 137 PC ae C40:6 Phosphatidylcholine acyl-alkyl C40:6 * 0.36 1000 138 PC ae C42:0 Phosphatidylcholine acyl-alkyl C42:0 * 1.55 1000 139 PC ae C42:1 Phosphatidylcholine acyl-alkyl C42:1 * 0.16 1000 140 PC ae C42:2 Phosphatidylcholine acyl-alkyl C42:2 * 0.07 1000 141 PC ae C42:3 Phosphatidylcholine acyl-alkyl C42:3 * 0.06 1000 142 PC ae C42:4 Phosphatidylcholine acyl-alkyl C42:4 * 0.21 1000 143 PC ae C42:5 Phosphatidylcholine acyl-alkyl C42:5 * 2.67 1000 144 PC ae C44:3 Phosphatidylcholine acyl-alkyl C44:3 * 0.03 1000 145 PC ae C44:4 Phosphatidylcholine acyl-alkyl C44:4 * 0.05 1000 146 PC ae C44:5 Phosphatidylcholine acyl-alkyl C44:5 * 0.19 1000 147 PC ae C44:6 Phosphatidylcholine acyl-alkyl C44:6 * 0.39 1000

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AbsoluteIDQ™ Kit

Table 1.5 Sphingolipids

Analyte Quantification Range MetIQ Short Name Biochemical Name

Quality Criterion Lower Limit (µM) Upper Limit (µM)

148 SM (OH) C14:1 Hydroxysphingomyeline C14:1 * 0.24 1000 149 SM C16:0 Sphingomyeline C16:0 * 3.39 1000 150 SM C16:1 Sphingomyeline C16:1 * 0.42 1000 151 SM (OH) C16:1 Hydroxysphingomyeline C16:1 * 0.19 1000 152 SM C18:0 Sphingomyeline C18:0 * 2.33 1000 153 SM C18:1 Sphingomyeline C18:1 * 0.43 1000 154 SM C20:2 Sphingomyeline C20:2 * 0.37 1000 155 SM C22:3 Sphingomyeline C22:3 * 4.02 1000 156 SM (OH) C22:1 Hydroxysphingomyeline C22:1 * 0.73 1000 157 SM (OH) C22:2 Hydroxysphingomyeline C22:2 * 0.64 1000 158 SM C24:0 Sphingomyeline C24:0 * 1.16 1000 159 SM C24:1 Sphingomyeline C24:1 * 3.07 1000 160 SM (OH) C24:1 Hydroxysphingomyeline C24:1 * 0.21 1000 161 SM C26:0 Sphingomyeline C26:0 * 0.41 1000 162 SM C26:1 Sphingomyeline C26:1 * 0.11 1000

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Specificity

The analytes were tested for selectivity and specificity in case there were corresponding standards available. In the following tables two criteria are listed: i) the effects of a single analyte/standard on other MRM pairs with respect to the influence on blank values, ii) the intensities of analyte-derived peaks other than the expected one (the expected analyte peak was set to 100% and additional peak intensities are only listed above 15%). All data show mean values of three replicate tests. In Table 2, all potential interferences are listed that could appear during mass spectrometric data acquisition. Table 2: Potential Interferences

Analyte Effects on other MRM pairs Other Analyte Peak Intensities

Arg 15N2-Arg >10 × blank Gln Trp > 10 × blank D5-Gln D3 Met > 10 × blank

lysoPC a C9:0 (PC aa C34:3, PC aa C36:3) > 10 × blank

Met D5-Gln > 10 × blank

PC aa C28:0 (lysoPC a C14:0, PC aa C26;0, PC ae C30:0, PC aa C30:0, PC ae C40:3, PC ae C40:1, PC ae C 42:3) > 10 × blank

PC aa C40:0

(PC aa C30:0, PC ae C36:0, PC aa C 36:6, PC aa C 36:0, PC ae C40:6, PC ae C40:0, PC ae C42:5, PC ae C42:2, PC ae C 42:0, PC aa C42:6, PC aa C42:5, PC aa C 42:0, PC ae C44:6, PC ae C44:5, PC ae C44:3) > 10 × blank

PC aa C40:0

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AbsoluteIDQ™ Kit

Analyte Effects on other MRM pairs Other Analyte Peak Intensities

PC aa C40:0

(PC aa C30:0, PC ae C36:0, PC aa C 36:6, PC aa C 36:0, PC ae C40:6, PC ae C40:0, PC ae C42:5, PC ae C42:2, PC ae C 42:0, PC aa C42:6, PC aa C42:5, PC aa C 42:0, PC ae C44:6, PC ae C44:5, PC ae C44:3) > 10 × blank

PC aa C36:0

(lysoPC a C18:0, PC aa C 36:0, PC aa C 36:6, PC ae C38:6, PC ae C38:5, PC ae C38:4, PC ae C38:0, PC ae C42:3, PC ae C44:4, PC ae C44:3) > 10 × blank

PC aa C24:0 (lysoPC a C24:0, lysoPC a C26:1, PC ae C36:1, PC ae C42:5) > 10 × blank

SM C6:0

(lysoPC a C24:0, lysoPC a C26:0, lysoPC a C26:1, PC ae C28:1, lysoPC a C28:0, PC aa C28:0, PC aa C30:0, PC aa C32:1) > 10 × blank

SM C18:0 (SM OH C14:1, SM C18:1, PC aa C32:2, PC aa C32:1, PC aa C32:0,PC ae C34:1) > 10 × blank PC aa C 32:1 40%

Thr PC aa C28:1 25%

Analytical Specifications Page 13

Linearity, Precision and Accuracy

The linearity of the assay was determined by analyzing extracts produced with the Kit after performing the assay procedure with 29 different standard chemicals at six different concentration levels. The standards represented 12 acylcarnitines, 12 amino acids, one hexose, one lyso- and two phospholipids, and one sphingolipid. A linear regression was performed using data of six repetitive preparations by plotting the mean concentrations of the replicates against the theoretical amount. R2 values were calculated whereby all analytes exhibited values of R2 ≥ 0.990. Moreover, for each standard the precision and accuracy were determined at the different concentration levels tested. In nearly all cases the precision was well below 10%, in a few cases it lay in the range from 10 – 20%. In case of the accuracy a deviation inside the 70 – 130% range was defined as valid. Some analytes did not meet this criterion: values for Arginine-PTC and Glutamine-PTC standards were inaccurate above concentrations of 5µM and 20µM, respectively. Further tests were performed to determine the accuracy in dependance of the matrix although this was not possible for all analyte classes since their concentration values in plasma are very low. Three levels of concentrations were spiked into pooled human plasma and measured in replicates of six. In addition to the two analytes mentioned before, it was observed that Tyrosine-PTC did not fall in the range of validity (80 – 120% of theoretical concentration). Therefore this analyte was also tagged with two asterisks in Table 1.2. The values for the precision were all valid, namely below 15% deviation from the theoretical values.

Functional Sensitivity

A substantial part of the metabolites that are determined by the AbsoluteIDQ Kit are often below the limit of detection in normal blood plasma. However, since it is well known that metabolite concentrations may change dramatically in disease states or upon external influences, it is important to measure those as well. Thus, if appropriate, the functional sensitivity was set to five times the value of the zero sample.

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AbsoluteIDQ™ Kit

With respect to the upper limit, the values were set by either taking the upper limit of the linearity range or by setting an artificial value in case of suitable standards were not available. The quantification ranges of the Kit are listed in Tables 1.1 – 1.5.

Reproducibility

The reproducibility of the Kit was determined by assaying three spiking levels of blood plasma (low, medium, high) using the available standards with n=6 replicates. For acylcarnitines, aminoacids and hexose the coefficients of variance (CVs) were all below 15% and therefore met the criteria provided in FDA Guidance for Industry - Bioanalytical Method Validation. For glycerophospho- and sphingolipids, the CVs were all below 20%. Moreover, intensive field testing was performed and confirmed both inter-labe and intra-lab reproducibilities.

Stability

The performance of the AbsoluteIDQ Kit is based on the functionality and integrity of all components. BIOCRATES guarantees the proper performance of the Kit plate for three months upon delivery if stored unopened in the original plastic bag (the Kit plate bag is sealed under nitrogen) and away from light at 4°C. Additionally, there are two more important stability criteria: the on-board stability and the autosampler stability. The on-board stability is defined as the time period in which the Kit plate can be processed after the sample material has been added without significant loss of analytical performance. On-board stability for the Kit is 12 hours if stored at 4°C and away from strong light. The autosampler stability is 48 hours at 10°C. This is the time in which the final extracts can be kept in the deep-well plate before MS analysis without significant loss of analytical performance.