NeoBase 2 Non-derivatized MSMS Kit

{0} # 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY ASSAY ONLY TEMPLATE A. 510(k) Number: k173568 B. Purpose for Submission: New Device C. Measurand: Amino acids, free carnitine, acylcarnitines, succinylacetone, nucleotides and lysophospholipids D. Type of Test: Quantitative measurement by mass spectrometry E. Applicant: Wallac Oy, a subsidiary of PerkinElmer Inc. F. Proprietary and Established Names: NeoBase 2 Non-derivatized MSMS kit G. Regulatory Information: 1. Regulation section: 21 CFR §862.1055 Newborn screening test system for amino acids, free carnitine, and acylcarnitines using tandem mass spectrometry 2. Classification: Class II 3. Product code: NQL 1 {1} #### 4. Panel: Chemistry (75) ### H. Intended Use: #### 1. Intended use(s): See Indications for use below. #### 2. Indication(s) for use: The NeoBase 2 Non-derivatized MSMS kit is intended for the measurement and evaluation of amino acid, succinylacetone, free carnitine, acylcarnitine, nucleoside and lysophospholipid concentrations (Table 1) with a tandem mass spectrometer from newborn heel prick blood specimens dried on filter paper. Quantitative analysis of these analytes and their relationship with each other is intended to provide analyte concentration profiles that may aid in screening newborns for metabolic disorders. Table 1. Analytes measured by the NeoBase2 Non-derivatized MSMS kit: | Analyte Name | Abbreviation | | --- | --- | | Amino acids | | | Alanine | Ala | | Arginine | Arg | | Argininosuccinic acid | Asa | | Citrulline | Cit | | Glutamine\Lysine (*) | Gln\Lys | | Glutamic acid | Glu | | Glycine | Gly | | Leucine\Isoleucine\Hydroxyproline (*) | Leu\Ile\Pro-OH | | Methionine | Met | | Ornithine | Orn | | Phenylalanine | Phe | | Proline | Pro | | Tyrosine | Tyr | | Valine | Val | | Carnitines | | | Free carnitine | C0 | | Acetylcarnitine | C2 | | Propionylcarnitine | C3 | | Malonylcarnitine\3-Hydroxy-butyrylcarnitine (*) | C3DC\C4OH | | Butyrylcarnitine | C4 | | Methylmalonyl\3-Hydroxy-isovalerylcarnitine (*) | C4DC\C5OH | | Isovalerylcarnitine | C5 | | Tiglylcarnitine | C5:1 | 2 {2} | Analyte Name | Abbreviation | | --- | --- | | Glutarylcarnitine\3-Hydroxy-hexanoylcarnitine (*) | C5DC\C6OH | | Hexanoylcarnitine | C6 | | Adipylcarnitine | C6DC | | Octanoylcarnitine | C8 | | Octenoylcarnitine | C8:1 | | Decanoylcarnitine | C10 | | Decenoylcarnitine | C10:1 | | Decadienoylcarnitine | C10:2 | | Dodecanoylcarnitine | C12 | | Dodecenoylcarnitine | C12:1 | | Tetradecanoylcarnitine (Myristoylcarnitine) | C14 | | Tetradecenoylcarnitine | C14:1 | | Tetradecadienoylcarnitine | C14:2 | | 3-Hydroxy-tetradecanoylcarnitine | C14OH | | Hexadecanoylcarnitine (Palmitoylcarnitine) | C16 | | Hexadecenoylcarnitine | C16:1 | | 3-Hydroxy-hexadecanoylcarnitine | C16OH | | 3-Hydroxy-hexadecenoylcarnitine\Heptadecanoylcarnitine (*) | C16:1OH\C17 | | Octadecanoylcarnitine (Stearoylcarnitine) | C18 | | Octadecenoylcarnitine (Oleylcarnitine) | C18:1 | | Octadecadienoylcarnitine (Linoleylcarnitine) | C18:2 | | 3-Hydroxy-octadecanoylcarnitine | C18OH | | 3-Hydroxy-octadecenoylcarnitine | C18:1OH | | 3-Hydroxy-octadecadienoylcarnitine | C18:2OH | | Ketones | | | Succinylacetone | SA | | Nucleosides | | | Adenosine | ADO | | 2'-deoxyadenosine | D-ADO | | Lysophospholipids | | | C24:0 lysophosphatidylcholine | C24:0-LPC | | C26:0 lysophosphatidylcholine | C26:0-LPC | (*) Analytes in these rows are either isomers or isobars and cannot be distinguished in the tandem mass spectrometry experiment. ### 3. Special conditions for use statement(s): For prescription use. The NeoBase2 Non-derivatized MSMS Kit is a screening assay, not intended for confirmatory or prenatal testing. As with any other in vitro screening test, the data obtained using this kit should be used as an aid to other medically established procedures 3 {3} and results interpreted in conjunction with other clinical data available to the clinician. A diagnostic procedure should be used for confirmation of presumptive abnormal amino acid, succinylacetone, free carnitine, acylcarnitine, nucleoside and lysophospholipid profiles. Users should follow local guidelines for follow-up and confirmatory testing. # 4. Special instrument requirements: Perkin Elmer TQD MS/MS Newborn Screening System. # I. Device Description: Each NeoBase 2 Non-derivatized MSMS kit contains reagents for 960 assays. The kit is designed to be used with NeoBase 2 Non-derivatized Assay Solutions consisting of Neo MSMS Flow Solvent and NeoBase 2 Extraction Solution and NeoBase 2 Succinylacetone Assay Solution. - NeoBase 2 Internal Standards - NeoBase 2 Controls Low, High - 3 filter paper cassettes (Whatman, no. 903) containing 3 spots of each level per cassette - Microplate, U-bottomed - 20 plates - Adhesive microplate covers - 20 sheets - Barcode labels for the plates - 30 pcs (10 different barcodes, 3 pcs of each) - Lot-specific quality control certificate This kit contains components manufactured from human blood. The source materials have been tested by FDA-approved methods for hepatitis B surface antigen, anti-hepatitis C and anti-HIV 1 and 2 antibodies and found to be negative. # J. Substantial Equivalence Information: # 1. Predicate device name(s): NeoBase Non-derivatized MSMS kit # 2. Predicate 510(k) number(s): k093916 4 {4} # 3. Comparison with predicate: | Similarities | | | | --- | --- | --- | | Item | Device | Predicate | | Intended Use | The NeoBase2 Non-derivatized MSMS kit is intended for the measurement and evaluation of amino acid, succinylacetone, free carnitine, acylcarnitine concentrations with a tandem mass spectrometer from newborn heel prick blood specimens dried on filter paper. | Same | | Disorders screened | Amino-, organic-, and fatty acid metabolic disorders | Same | | Test Principle | Analytes in sample are measured by tandem mass spectrometry through analyte-specific mass transitions appropriate for each type of analyte. The extracted analytes are measured for set time periods and compared to the signal intensities produced by the corresponding isotope-labeled internal standards. The concentrations are determined by comparing the signal intensities of the known standards to the measured analytes. | Same | | Calibrators | Internal calibration using several isotopically labeled standards, included as dried material in vials. Internal standards must be reconstituted with extraction solution prior to their use. | Same | | Assay format | Non-derivatized (analytes measured in their native | Same | 5 {5} | Similarities | | | | --- | --- | --- | | Item | Device | Predicate | | | forms) | | | Differences | | | | --- | --- | --- | | Item | Device | Predicate | | Analytes Measured | Amino acids, free carnitine, acylcarnitines, succinylacetone, nucleosides, and lysophospholipids | Amino acids, free carnitine, acylcarnitines, and succinylacetone | | MSMS Instrument Flow Solvent | 84% Acetonitrile 16% water 0.1% Formic Acid | 78% Methanol, 22% water, 3mM oxalic acid | # **K. Standard/Guidance Document Referenced (if applicable):** Clinical and Laboratory Standards Institute (CLSI) EP5-A3 Evaluation of Precision of Quantitative Measurement Procedures; Approved Guideline—Third Edition CLSI EP06-A Evaluation of the Linearity of Quantitative Measurement Procedures: A Statistical Approach; Approved Guideline CLSI EP7-A2 Interference Testing in Clinical Chemistry; Approved Guideline—Second Edition # **L. Test Principle:** The measurement of amino acids, succinylacetone, free carnitine, acylcarnitines, nucleosides, and lysophospholipids with the NeoBase 2 assay involves extraction of dried blood spots from newborns with a solution containing stable-isotope labeled internal standards and analysis using a tandem mass spectrometry (MSMS) system. The response of each analyte relative to their corresponding stable-isotope labeled internal standard is proportional to the analyte concentration. In the NeoBase 2 Non-derivatized MSMS Kit, data is acquired in the Multiple Reaction Monitoring (MRM) mode. During this acquisition, a collisionally induced product of each analyte is measured for a set time period. Data acquisition and processing is performed by the software package included with the system. The triple-quadrupole mass spectrometer that is used for these measurements is a computer-controlled device that separates and quantitates ions based on their mass to charge (m/z) ratio. The extracted sample is delivered to the ion source of the mass spectrometer by the liquid chromatography (LC) system consisting of the autosampler, micro pump(s) and solvent vacuum degasser. What is reported in the MSMS MRM spectrum is the m/z value of the precursor ions that generated a desired product. Analyte extraction with the NeoBase 2 Non-derivatized MSMS assay is accomplished for the 6 {6} amino acids, carnitines, nucleosides and lysophospholipids by adding extraction working solution (EWS) containing NeoBase 2 Extraction Solution and NeoBase 2 Internal Standards to the sample during the incubation step. However, for the extraction and measurement of SA, the compound needs to be derivatized. This takes place simultaneously with the extraction of other analytes by addition of an aliquot of the NeoBase 2 Succinylacetone Assay Solution to the EWS. ### M. Performance Characteristics (if/when applicable): #### 1. Analytical performance: ##### a. Precision/Reproducibility: Three precision studies were conducted using dried blood spot punches following the recommendations in the CLSI guideline EP05-A3. The repeatability and within-laboratory variation for NeoBase 2 Non-derivatized MSMS kit were based on 80 determinations: 40 plates measured over 20 working days, each plate with 2 replicates per sample. The samples were measured with the TQD MSMS system. Between-lot variation is based on 75 determinations: 15 plates measured over five working days using three kit lots, each plate having 5 replicates per sample. One TQD was used. Between-instrument variation was based on 50 determinations. 10 plates were measured with two TQDs over 5 working days, each plate having 5 replicates per sample. The results are summarized below: Ala | Sample | Total mean μmol/L | Repeatability | | Within-lab | | Between-lot | | Between-instrument | | Total variation | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | | SD | CV % | SD | CV % | SD | CV % | SD | CV % | SD | CV % | | 1 | 141 | 5.7 | 4.3 | 8.9 | 6.7 | 4.1 | 2.8 | 7.6 | 5.4 | 12 | 8.8 | | 2 | 347 | 17 | 5.0 | 20 | 5.9 | 6.2 | 1.8 | 11 | 3.1 | 24 | 6.9 | | 3 | 407 | 16 | 3.9 | 18 | 4.4 | 4.2 | 1.0 | 18 | 4.4 | 26 | 6.4 | | 4 | 519 | 24 | 4.5 | 27 | 5.1 | 15 | 3.1 | 23 | 4.2 | 38 | 7.3 | | 5 | 2832 | 114 | 4.0 | 171 | 6.0 | 82 | 3.0 | 0.05 | <0.01 | 190 | 6.7 | Arg | Sample | Total mean μmol/L | Repeatability | | Within-lab | | Between-lot | | Between-instrument | | Total variation | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | | SD | CV % | SD | CV % | SD | CV % | SD | CV % | SD | CV % | | 1 | 7.5 | 0.49 | 6.1 | 0.85 | 11 | 0.47 | 6.8 | <0.01 | <0.01 | 0.97 | 13 | | 2 | 23 | 1.4 | 6.0 | 1.6 | 6.9 | 0.11 | 0.49 | 0.61 | 2.6 | 1.7 | 7.6 | | 3 | 67 | 2.8 | 4.2 | 3.5 | 5.2 | 1.7 | 2.6 | 0.57 | 0.85 | 3.9 | 5.8 | | 4 | 157 | 6.6 | 4.1 | 8.8 | 5.4 | 5.2 | 3.5 | 2.8 | 1.8 | 11 | 6.8 | | 5 | 1241 | 52 | 4.1 | 72 | 5.7 | 39 | 3.3 | <0.01 | <0.01 | 82 | 6.6 | 7 {7} Asa | Sample | Total mean μmol/L | Repeatability | | Within-lab | | Between-lot | | Between-instrument | | Total variation | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | | SD | CV % | SD | CV % | SD | CV % | SD | CV % | SD | CV % | | 1 | 0.30 | 0.05 | 22 | 0.06 | 26 | 0.01 | 1.8 | 0.07 | 29 | 0.10 | 32 | | 2 | 2.3 | 0.14 | 6.0 | 0.34 | 15 | 0.07 | 3.1 | 0.02 | 1.1 | 0.35 | 15 | | 3 | 8.4 | 0.53 | 6.2 | 1.0 | 12 | 0.44 | 5.3 | <0.01 | <0.01 | 1.1 | 13 | | 4 | 23 | 0.93 | 3.8 | 3.1 | 13 | 0.95 | 4.5 | <0.01 | 0.01 | 3.2 | 14 | | 5 | 50 | 1.8 | 3.4 | 7.0 | 13 | 2.1 | 4.6 | 0.19 | 0.38 | 7.3 | 15 | Cit | Sample | Total mean μmol/L | Repeatability | | Within-lab | | Between-lot | | Between-instrument | | Total variation | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | | SD | CV % | SD | CV % | SD | CV % | SD | CV % | SD | CV % | | 1 | 10 | 0.79 | 8.3 | 1.1 | 11 | 0.33 | 3.0 | 0.70 | 6.6 | 1.3 | 13 | | 2 | 69 | 3.9 | 5.8 | 4.9 | 7.2 | 2.1 | 3.1 | 1.7 | 2.4 | 5.6 | 8.2 | | 3 | 205 | 10 | 4.9 | 11 | 5.2 | 5.4 | 2.7 | 4.7 | 2.3 | 13 | 6.3 | | 4 | 480 | 27 | 5.5 | 33 | 6.8 | 11 | 2.3 | 12 | 2.5 | 37 | 7.6 | | 5 | 926 | 39 | 4.2 | 55 | 5.9 | 27 | 3.0 | 0.03 | <0.01 | 61 | 6.6 | Gln\Lys | Sample | Total mean μmol/L | Repeatability | | Within-lab | | Between-lot | | Between-instrument | | Total variation | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | | SD | CV % | SD | CV % | SD | CV % | SD | CV % | SD | CV % | | 1 | 40 | 2.2 | 5.5 | 3.1 | 7.8 | 1.0 | 2.5 | 2.2 | 5.4 | 3.9 | 9.8 | | 2 | 470 | 23 | 4.9 | 29 | 6.2 | 15 | 3.3 | 9.6 | 2.0 | 34 | 7.3 | | 3 | 654 | 26 | 3.9 | 30 | 4.6 | 3.3 | 0.52 | 13 | 2.0 | 33 | 5.0 | | 4 | 1043 | 47 | 4.4 | 56 | 5.3 | 37 | 3.7 | 27 | 2.6 | 72 | 6.9 | | 5 | 2264 | 84 | 3.7 | 106 | 4.7 | 20 | 0.90 | 41 | 1.8 | 116 | 5.1 | Gly | Sample | Total mean μmol/L | Repeatability | | Within-lab | | Between-lot | | Between-instrument | | Total variation | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | | SD | CV % | SD | CV % | SD | CV % | SD | CV % | SD | CV % | | 1 | 245 | 12 | 5.0 | 18 | 7.9 | 7.4 | 2.8 | 12 | 4.8 | 22 | 9.0 | | 2 | 322 | 20 | 6.6 | 21 | 7.0 | 6.6 | 1.9 | 18 | 5.7 | 28 | 8.8 | | 3 | 521 | 23 | 4.6 | 26 | 5.3 | 6.1 | 1.1 | 27 | 5.2 | 38 | 7.3 | | 4 | 928 | 44 | 5.0 | 58 | 6.5 | 26 | 2.8 | 50 | 5.3 | 81 | 8.7 | | 5 | 2752 | 128 | 4.8 | 171 | 6.4 | 77 | 2.7 | 95 | 3.4 | 210 | 7.6 | 8 {8} Leu\Ile\Pro-OH | Sample | Total mean μmol/L | Repeatability | | Within-lab | | Between-lot | | Between-instrument | | Total variation | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | | SD | CV % | SD | CV % | SD | CV % | SD | CV % | SD | CV % | | 1 | 58 | 2.0 | 3.7 | 3.3 | 6.1 | 0.54 | 0.87 | 3.6 | 6.1 | 4.9 | 8.4 | | 2 | 202 | 9.3 | 4.7 | 12 | 5.9 | 4.7 | 2.3 | 5.6 | 2.7 | 14 | 6.9 | | 3 | 353 | 12 | 3.4 | 16 | 4.4 | 2.7 | 0.80 | 7.6 | 2.1 | 18 | 5.0 | | 4 | 668 | 28 | 4.1 | 37 | 5.4 | 17 | 2.6 | 13 | 1.8 | 42 | 6.3 | | 5 | 1137 | 48 | 4.1 | 77 | 6.7 | 13 | 1.2 | 22 | 1.8 | 81 | 7.1 | Met | Sample | Total mean μmol/L | Repeatability | | Within-lab | | Between-lot | | Between-instrument | | Total variation | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | | SD | CV % | SD | CV % | SD | CV % | SD | CV % | SD | CV % | | 1 | 1.8 | 0.29 | 36 | 0.35 | 43 | 0.26 | 8.3 | 0.50 | 34 | 0.67 | 37 | | 2 | 50 | 3.0 | 6.3 | 3.9 | 8.2 | 1.9 | 3.7 | 1.9 | 3.7 | 4.8 | 9.5 | | 3 | 154 | 5.4 | 3.5 | 8.7 | 5.7 | 2.3 | 1.5 | 4.4 | 2.8 | 10 | 6.5 | | 4 | 373 | 18 | 4.7 | 25 | 6.6 | 9.8 | 2.7 | 9.1 | 2.4 | 28 | 7.6 | | 5 | 703 | 32 | 4.5 | 52 | 7.4 | 14 | 2.0 | 24 | 3.2 | 59 | 8.3 | Orn | Sample | Total mean μmol/L | Repeatability | | Within-lab | | Between-lot | | Between-instrument | | Total variation | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | | SD | CV % | SD | CV % | SD | CV % | SD | CV % | SD | CV % | | 1 | 30 | 3.4 | 11 | 3.7 | 12 | 0.50 | 1.7 | <0.01 | <0.01 | 3.7 | 12 | | 2 | 110 | 5.2 | 4.6 | 6.1 | 5.4 | 1.5 | 1.4 | <0.01 | <0.01 | 6.3 | 5.7 | | 3 | 202 | 7.8 | 3.7 | 9.9 | 4.7 | 2.3 | 1.2 | <0.01 | <0.01 | 10 | 5.0 | | 4 | 390 | 14 | 3.5 | 20 | 5.0 | 9.9 | 2.7 | <0.01 | <0.01 | 23 | 5.8 | | 5 | 1305 | 43 | 3.2 | 86 | 6.3 | 18 | 1.5 | 0.12 | 0.01 | 88 | 6.7 | Phe | Sample | Total mean μmol/L | Repeatability | | Within-lab | | Between-lot | | Between-instrument | | Total variation | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | | SD | CV % | SD | CV % | SD | CV % | SD | CV % | SD | CV % | | 1 | 22 | 1.1 | 5.3 | 1.5 | 7.2 | 0.43 | 1.7 | 1.1 | 4.8 | 1.9 | 8.3 | | 2 | 128 | 6.0 | 4.8 | 7.4 | 5.9 | 3.5 | 2.7 | 2.1 | 1.6 | 8.5 | 6.6 | | 3 | 343 | 11 | 3.3 | 15 | 4.3 | 4.7 | 1.4 | 0.04 | 0.01 | 16 | 4.5 | | 4 | 790 | 37 | 4.6 | 43 | 5.4 | 29 | 3.8 | 2.8 | 0.35 | 52 | 6.6 | | 5 | 1468 | 66 | 4.5 | 98 | 6.6 | 17 | 1.2 | 0.34 | 0.02 | 99 | 6.8 | 9 {9} Pro | Sample | Total mean μmol/L | Repeatability | | Within-lab | | Between-lot | | Between-instrument | | Total variation | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | | SD | CV % | SD | CV % | SD | CV % | SD | CV % | SD | CV % | | 1 | 40 | 1.8 | 4.6 | 2.7 | 6.9 | 0.57 | 1.4 | 1.6 | 3.9 | 3.1 | 7.8 | | 2 | 180 | 7.8 | 4.3 | 11 | 5.8 | 4.6 | 2.6 | 1.9 | 1.0 | 12 | 6.5 | | 3 | 318 | 11 | 3.4 | 15 | 4.7 | 2.6 | 0.86 | 0.04 | 0.01 | 16 | 4.9 | | 4 | 606 | 27 | 4.2 | 31 | 5.0 | 16 | 2.8 | <0.01 | <0.01 | 35 | 5.8 | | 5 | 1467 | 50 | 3.3 | 87 | 5.8 | 36 | 2.6 | 0.04 | <0.01 | 95 | 6.4 | Tyr | Sample | Total mean μmol/L | Repeatability | | Within-lab | | Between-lot | | Between-instrument | | Total variation | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | | SD | CV % | SD | CV % | SD | CV % | SD | CV % | SD | CV % | | 1 | 20 | 1.2 | 6.3 | 1.8 | 8.9 | 0.52 | 2.6 | 0.65 | 3.1 | 1.9 | 9.6 | | 2 | 110 | 4.8 | 4.4 | 6.2 | 5.7 | 3.6 | 3.3 | 1.1 | 0.99 | 7.3 | 6.6 | | 3 | 268 | 10 | 3.8 | 12 | 4.5 | 4.1 | 1.6 | 2.4 | 0.89 | 13 | 4.9 | | 4 | 596 | 28 | 4.7 | 33 | 5.5 | 15 | 2.6 | 0.01 | <0.01 | 36 | 6.1 | | 5 | 1585 | 67 | 4.1 | 90 | 5.6 | 40 | 2.6 | 0.02 | <0.01 | 99 | 6.2 | Val | Sample | Total mean μmol/L | Repeatability | | Within-lab | | Between-lot | | Between-instrument | | Total variation | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | | SD | CV % | SD | CV % | SD | CV % | SD | CV % | SD | CV % | | 1 | 57 | 2.2 | 4.2 | 3.3 | 6.3 | 0.81 | 1.3 | 3.0 | 5.3 | 4.5 | 8.0 | | 2 | 209 | 9.8 | 4.6 | 13 | 6.0 | 5.6 | 2.8 | 2.4 | 1.1 | 14 | 6.7 | | 3 | 321 | 9.7 | 2.9 | 14 | 4.4 | 4.2 | 1.4 | 2.2 | 0.67 | 15 | 4.7 | | 4 | 554 | 24 | 4.2 | 31 | 5.4 | 15 | 3.0 | <0.01 | <0.01 | 35 | 6.3 | | 5 | 1160 | 41 | 3.4 | 69 | 5.7 | 35 | 3.2 | 0.01 | <0.01 | 78 | 6.7 | C0 | Sample | Total mean μmol/L | Repeatability | | Within-lab | | Between-lot | | Between-instrument | | Total variation | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | | SD | CV % | SD | CV % | SD | CV % | SD | CV % | SD | CV % | | 1 | 8.6 | 0.47 | 5.6 | 0.57 | 6.8 | 0.04 | 0.47 | 0.47 | 5.3 | 0.74 | 8.5 | | 2 | 42 | 2.0 | 4.9 | 2.4 | 5.9 | 1.5 | 3.7 | 1.5 | 3.5 | 3.2 | 7.8 | | 3 | 90 | 3.1 | 3.4 | 4.2 | 4.7 | 1.1 | 1.2 | 2.2 | 2.4 | 4.9 | 5.4 | | 4 | 189 | 8.7 | 4.5 | 13 | 6.6 | 6.7 | 3.7 | 5.3 | 2.8 | 15 | 8.1 | | 5 | 1236 | 59 | 4.7 | 79 | 6.3 | 31 | 2.6 | 0.02 | <0.01 | 84 | 6.8 | 10 {10} C2 | Sample | Total mean μmol/L | Repeatability | | Within-lab | | Between-lot | | Between-instrument | | Total variation | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | | SD | CV % | SD | CV % | SD | CV % | SD | CV % | SD | CV % | | 1 | 3.6 | 0.15 | 4.4 | 0.25 | 7.2 | 0.09 | 2.5 | 0.09 | 2.6 | 0.28 | 7.9 | | 2 | 12 | 0.63 | 5.2 | 0.76 | 6.3 | 0.47 | 3.9 | 0.29 | 2.3 | 0.94 | 7.7 | | 3 | 19 | 0.62 | 3.4 | 0.81 | 4.4 | 0.09 | 0.49 | 0.51 | 2.7 | 0.97 | 5.2 | | 4 | 31 | 1.4 | 4.5 | 1.8 | 5.9 | 0.66 | 2.2 | 1.1 | 3.6 | 2.2 | 7.3 | | 5 | 328 | 13 | 4.1 | 20 | 6.1 | 11 | 3.3 | <0.01 | <0.01 | 23 | 6.9 | C3 | Sample | Total mean μmol/L | Repeatability | | Within-lab | | Between-lot | | Between-instrument | | Total variation | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | | SD | CV % | SD | CV % | SD | CV % | SD | CV % | SD | CV % | | 1 | 0.43 | 0.03 | 7.5 | 0.04 | 9.5 | 0.01 | 2.9 | 0.03 | 5.8 | 0.05 | 11 | | 2 | 4.4 | 0.22 | 5.2 | 0.27 | 6.3 | 0.14 | 3.2 | 0.22 | 4.7 | 0.37 | 8.5 | | 3 | 13 | 0.51 | 3.9 | 0.64 | 5.0 | 0.16 | 1.2 | 0.55 | 4.1 | 0.86 | 6.6 | | 4 | 31 | 1.6 | 5.3 | 2.1 | 6.8 | 1.2 | 4.0 | 1.4 | 4.3 | 2.8 | 9.0 | | 5 | 58 | 2.5 | 4.4 | 4.5 | 7.8 | 0.93 | 1.7 | 2.6 | 4.3 | 5.2 | 9.1 | C4 | Sample | Total mean μmol/L | Repeatability | | Within-lab | | Between-lot | | Between-instrument | | Total variation | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | | SD | CV % | SD | CV % | SD | CV % | SD | CV % | SD | CV % | | 1 | 0.06 | 0.01 | 11 | 0.01 | 12 | <0.01 | 3.5 | 0.01 | 13 | 0.01 | 17 | | 2 | 0.57 | 0.03 | 5.8 | 0.04 | 6.8 | 0.02 | 2.9 | 0.02 | 3.4 | 0.05 | 8.0 | | 3 | 1.8 | 0.05 | 3.1 | 0.09 | 5.2 | 0.01 | 0.31 | 0.04 | 2.2 | 0.10 | 5.7 | | 4 | 4.3 | 0.19 | 4.5 | 0.27 | 6.2 | 0.08 | 2.0 | 0.14 | 3.2 | 0.31 | 7.4 | | 5 | 20 | 0.89 | 4.4 | 1.2 | 5.9 | 0.54 | 2.7 | <0.01 | <0.01 | 1.3 | 6.5 | C5 | Sample | Total mean μmol/L | Repeatability | | Within-lab | | Between-lot | | Between-instrument | | Total variation | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | | SD | CV % | SD | CV % | SD | CV % | SD | CV % | SD | CV % | | 1 | 0.04 | 0.01 | 23 | 0.01 | 24 | <0.01 | 2.6 | <0.01 | 8.6 | <0.01 | 23 | | 2 | 1.0 | 0.05 | 5.3 | 0.06 | 6.3 | 0.03 | 2.8 | 0.02 | 2.3 | 0.07 | 7.2 | | 3 | 3.7 | 0.13 | 3.6 | 0.18 | 5.0 | 0.04 | 1.1 | 0.09 | 2.5 | 0.21 | 5.7 | | 4 | 9.2 | 0.42 | 4.6 | 0.57 | 6.2 | 0.31 | 3.6 | 0.23 | 2.4 | 0.69 | 7.5 | | 5 | 20 | 0.74 | 3.8 | 1.1 | 5.8 | 0.22 | 1.2 | <0.01 | <0.01 | 1.2 | 6.0 | 11 {11} C5DC\C6OH | Sample | Total mean μmol/L | Repeatability | | Within-lab | | Between-lot | | Between-instrument | | Total variation | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | | SD | CV % | SD | CV % | SD | CV % | SD | CV % | SD | CV% | | 1 | 0.04 | 0.01 | 22 | 0.01 | 23 | <0.01 | 9.2 | <0.01 | 15 | 0.01 | 27 | | 2 | 0.44 | 0.03 | 6.5 | 0.03 | 7.4 | 0.01 | 3.1 | 0.01 | 3.2 | 0.04 | 8.5 | | 3 | 1.5 | 0.08 | 5.4 | 0.10 | 6.6 | 0.02 | 1.1 | 0.08 | 5.0 | 0.13 | 8.3 | | 4 | 3.8 | 0.23 | 6.1 | 0.26 | 6.9 | 0.07 | 1.9 | 0.11 | 2.9 | 0.30 | 7.8 | | 5 | 35 | 2.0 | 5.8 | 2.4 | 6.9 | 1.1 | 3.1 | <0.01 | <0.01 | 2.7 | 7.6 | C6 | Sample | Total mean μmol/L | Repeatability | | Within-lab | | Between-lot | | Between-instrument | | Total variation | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | | SD | CV % | SD | CV % | SD | CV % | SD | CV % | SD | CV % | | 1 | 0.02 | <0.01 | 35 | <0.01 | 35 | <0.01 | 15 | <0.01 | 22 | <0.01 | 41 | | 2 | 0.38 | 0.02 | 6.1 | 0.03 | 7.1 | <0.01 | 2.4 | 0.02 | 5.7 | 0.04 | 9.3 | | 3 | 1.4 | 0.04 | 3.2 | 0.07 | 5.2 | 0.03 | 1.8 | 0.05 | 3.6 | 0.09 | 6.5 | | 4 | 3.5 | 0.16 | 4.5 | 0.20 | 5.7 | 0.10 | 3.1 | 0.13 | 3.7 | 0.26 | 7.5 | | 5 | 22 | 0.97 | 4.3 | 1.3 | 5.6 | 0.73 | 3.3 | <0.01 | 0.01 | 1.5 | 6.5 | C8 | Sample | Total mean μmol/L | Repeatability | | Within-lab | | Between-lot | | Between-instrument | | Total variation | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | | SD | CV % | SD | CV % | SD | CV % | SD | CV % | SD | CV % | | 1 | 0.03 | 0.01 | 26 | 0.01 | 30 | <0.01 | 16 | <0.01 | 8.3 | 0.01 | 36 | | 2 | 2.1 | 0.11 | 5.2 | 0.13 | 6.6 | 0.08 | 3.8 | 0.06 | 3.0 | 0.17 | 8.1 | | 3 | 7.6 | 0.30 | 3.9 | 0.35 | 4.6 | 0.12 | 1.6 | 0.12 | 1.6 | 0.39 | 5.1 | | 4 | 19 | 1.0 | 5.2 | 1.2 | 6.1 | 0.64 | 3.6 | 0.31 | 1.5 | 1.4 | 7.3 | | 5 | 36 | 1.8 | 5.0 | 2.6 | 7.1 | 0.44 | 1.3 | 0.71 | 1.9 | 2.7 | 7.5 | C10 | Sample | Total mean μmol/L | Repeatability | | Within-lab | | Between-lot | | Between-instrument | | Total variation | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | | SD | CV % | SD | CV % | SD | CV % | SD | CV % | SD | CV % | | 1 | 0.03 | <0.01 | 18 | 0.01 | 23 | <0.01 | 13 | <0.01 | 10 | <0.01 | 28 | | 2 | 0.45 | 0.02 | 5.7 | 0.03 | 6.6 | 0.01 | 2.4 | 0.03 | 6.0 | 0.04 | 9.1 | | 3 | 1.5 | 0.06 | 4.2 | 0.07 | 4.9 | 0.02 | 1.2 | 0.10 | 6.2 | 0.12 | 8.0 | | 4 | 3.9 | 0.21 | 5.5 | 0.25 | 6.6 | 0.11 | 2.9 | 0.23 | 5.6 | 0.35 | 9.2 | | 5 | 20 | 0.78 | 4.0 | 1.1 | 5.6 | 0.72 | 3.6 | 0.79 | 3.9 | 1.5 | 7.6 | 12 {12} C12 | Sample | Total mean μmol/L | Repeatability | | Within-lab | | Between-lot | | Between-instrument | | Total variation | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | | SD | CV % | SD | CV % | SD | CV % | SD | CV % | SD | CV % | | 1 | 0.02 | <0.01 | 24 | 0.01 | 61 | <0.01 | 5.2 | <0.01 | 17 | 0.01 | 70 | | 2 | 0.49 | 0.03 | 6.6 | 0.04 | 7.4 | 0.02 | 3.4 | <0.01 | 0.73 | 0.04 | 8.0 | | 3 | 1.8 | 0.07 | 4.0 | 0.08 | 4.8 | 0.01 | 0.76 | 0.05 | 2.7 | 0.10 | 5.5 | | 4 | 4.4 | 0.24 | 5.4 | 0.27 | 6.2 | 0.16 | 3.7 | 0.09 | 1.9 | 0.33 | 7.4 | | 5 | 20 | 0.67 | 3.3 | 1.1 | 5.3 | 0.49 | 2.4 | <0.01 | 0.01 | 1.2 | 5.8 | C14 | Sample | Total mean μmol/L | Repeatability | | Within-lab | | Between-lot | | Between-instrument | | Total variation | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | | SD | CV % | SD | CV % | SD | CV % | SD | CV% | SD | CV% | | 1 | 0.05 | 0.01 | 8.3 | 0.02 | 38 | <0.01 | 3.5 | <0.01 | 1.6 | 0.02 | 43 | | 2 | 0.57 | 0.03 | 5.7 | 0.04 | 7.3 | 0.01 | 1.6 | <0.01 | 0.03 | 0.04 | 7.5 | | 3 | 1.8 | 0.07 | 3.8 | 0.08 | 4.4 | 0.03 | 1.6 | <0.01 | 0.01 | 0.09 | 4.7 | | 4 | 4.3 | 0.22 | 5.1 | 0.25 | 5.7 | 0.12 | 2.8 | <0.01 | 0.01 | 0.28 | 6.4 | | 5 | 19 | 0.66 | 3.5 | 1.0 | 5.4 | 0.43 | 2.3 | <0.01 | <0.01 | 1.1 | 5.9 | C16 | Sample | Total mean μmol/L | Repeatability | | Within-lab | | Between-lot | | Between-instrument | | Total variation | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | | SD | CV % | SD | CV % | SD | CV % | SD | CV % | SD | CV % | | 1 | 1.0 | 0.05 | 4.9 | 0.08 | 7.8 | 0.02 | 2.3 | 0.04 | 3.8 | 0.09 | 8.9 | | 2 | 3.7 | 0.17 | 4.6 | 0.18 | 5.1 | 0.13 | 3.6 | 0.13 | 3.3 | 0.26 | 7.0 | | 3 | 11 | 0.42 | 3.9 | 0.51 | 4.7 | 0.07 | 0.62 | 0.23 | 2.1 | 0.56 | 5.2 | | 4 | 25 | 1.1 | 4.6 | 1.5 | 6.1 | 0.35 | 1.5 | 0.61 | 2.4 | 1.6 | 6.7 | | 5 | 60 | 2.0 | 3.4 | 3.2 | 5.3 | 1.3 | 2.2 | 1.1 | 1.8 | 3.6 | 6.0 | C18 | Sample | Total mean μmol/L | Repeatability | | Within-lab | | Between-lot | | Between-instrument | | Total variation | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | | SD | CV % | SD | CV % | SD | CV % | SD | CV% | SD | CV % | | 1 | 0.51 | 0.02 | 4.3 | 0.05 | 10 | 0.010 | 2.0 | <0.01 | 0.01 | 0.05 | 11 | | 2 | 1.3 | 0.06 | 4.8 | 0.09 | 6.8 | 0.03 | 2.7 | <0.01 | <0.01 | 0.10 | 7.4 | | 3 | 3.0 | 0.10 | 3.3 | 0.14 | 4.7 | 0.03 | 1.0 | <0.01 | <0.01 | 0.15 | 4.9 | | 4 | 6.3 | 0.25 | 3.8 | 0.29 | 4.4 | 0.03 | 0.58 | <0.01 | <0.01 | 0.29 | 4.6 | | 5 | 38 | 1.4 | 3.5 | 2.0 | 5.2 | 0.79 | 2.2 | <0.01 | <0.01 | 2.2 | 5.7 | 13 {13} SA | Sample | Total mean μmol/L | Repeatability | | Within-lab | | Between-lot | | Between-instrument | | Total variation | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | | SD | CV % | SD | CV % | SD | CV % | SD | CV % | SD | CV % | | 1 | 0.39 | 0.04 | 20 | 0.04 | 23 | 0.02 | 3.2 | 0.16 | 47 | 0.17 | 43 | | 2 | 3.6 | 0.32 | 9.0 | 0.62 | 18 | 0.09 | 2.4 | <0.01 | <0.01 | 0.63 | 17 | | 3 | 13 | 0.79 | 5.8 | 2.3 | 17 | 0.22 | 1.6 | <0.01 | 0.01 | 2.3 | 17 | | 4 | 37 | 2.1 | 5.6 | 6.4 | 17 | 0.77 | 2.2 | <0.01 | <0.01 | 6.5 | 17 | | 5 | 75 | 5.1 | 6.4 | 14 | 18 | 0.15 | 0.22 | 0.03 | 0.04 | 14 | 19 | ADO | Sample | Total mean μmol/L | Repeatability | | Within-lab | | Between-lot | | Between-instrument | | Total variation | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | | SD | CV % | SD | CV % | SD | CV % | SD | CV % | SD | CV % | | 1 | 0.15 | 0.02 | 14 | 0.02 | 16 | <0.01 | 2.1 | 0.02 | 17 | 0.03 | 22 | | 2 | 1.4 | 0.11 | 8.3 | 0.13 | 9.3 | 0.02 | 1.3 | 0.04 | 2.8 | 0.13 | 9.9 | | 3 | 5.5 | 0.23 | 4.1 | 0.28 | 5.0 | 0.07 | 1.2 | 0.08 | 1.4 | 0.29 | 5.3 | | 4 | 15 | 0.70 | 4.7 | 0.83 | 5.5 | 0.23 | 1.6 | 0.15 | 1.0 | 0.87 | 6.0 | | 5 | 29 | 1.0 | 3.4 | 1.8 | 6.2 | 0.43 | 1.5 | <0.01 | 0.01 | 1.9 | 6.4 | C26:0-LPC | Sample | Total mean μmol/L | Repeatability | | Within-lab | | Between-lot | | Between-instrument | | Total variation | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | | SD | CV % | SD | CV % | SD | CV % | SD | CV % | SD | CV % | | 1 | 0.22 | 0.03 | 13 | 0.03 | 13 | 0.01 | 4.5 | <0.01 | 0.02 | 0.03 | 13 | | 2 | 0.69 | 0.06 | 9.2 | 0.08 | 11 | 0.01 | 1.1 | 0.02 | 2.8 | 0.08 | 11 | | 3 | 1.4 | 0.11 | 7.4 | 0.13 | 8.4 | 0.01 | 0.47 | 0.11 | 8.2 | 0.17 | 12 | | 4 | 2.9 | 0.15 | 4.9 | 0.21 | 6.8 | 0.01 | 0.55 | 0.18 | 6.3 | 0.28 | 9.7 | | 5 | 5.5 | 0.34 | 5.6 | 0.43 | 7.1 | 0.10 | 2.1 | 0.49 | 8.8 | 0.66 | 12 | The reproducibility of the NeoBase2 Non-derivatized MSMS assay was determined in an additional study on the TQD MSMS Screening System across 2 external sites and one internal site. The reproducibility is based on 75 determinations: in each laboratory 5 plates were measured over 5 working days using one kit lot and each plate having 5 replicates per sample. The results are summarized below: 14 {14} Ala | Sample | Total mean μmol/L | Within-lab | | Between-lab | | Reproducibility | | | --- | --- | --- | --- | --- | --- | --- | --- | | | | SD | CV% | SD | CV% | SD | CV% | | 1 | 336 | 19 | 5.9 | 25 | 7.5 | 32 | 9.5 | | 2 | 430 | 23 | 5.5 | 38 | 8.9 | 45 | 10 | | 3 | 693 | 38 | 5.6 | 71 | 10 | 81 | 11 | Arg | Sample | Total mean μmol/L | Within-lab | | Between-lab | | Reproducibility | | | --- | --- | --- | --- | --- | --- | --- | --- | | | | SD | CV% | SD | CV% | SD | CV% | | 1 | 8.2 | 1.1 | 13 | 0.69 | 8.4 | 1.3 | 15 | | 2 | 49 | 2.4 | 4.8 | 2.0 | 4.1 | 3.1 | 6.3 | | 3 | 175 | 9.0 | 5.1 | 6.5 | 3.7 | 11 | 6.3 | Asa | Sample | Total mean μmol/L | Within-lab | | Between-lab | | Reproducibility | | | --- | --- | --- | --- | --- | --- | --- | --- | | | | SD | CV% | SD | CV% | SD | CV% | | 1 | 0.53 | 0.16 | 29 | 0.36 | 67 | 0.39 | 73 | | 2 | 6.7 | 0.54 | 8.1 | 0.11 | 1.7 | 0.55 | 8.3 | | 3 | 29 | 1.9 | 6.4 | 1.6 | 5.3 | 2.5 | 8.3 | Cit | Sample | Total mean μmol/L | Within-lab | | Between-lab | | Reproducibility | | | --- | --- | --- | --- | --- | --- | --- | --- | | | | SD | CV% | SD | CV% | SD | CV% | | 1 | 21 | 2.3 | 10 | 0.38 | 1.8 | 2.4 | 11 | | 2 | 91 | 6.5 | 7.1 | 0.88 | 0.97 | 6.5 | 7.2 | | 3 | 292 | 19 | 6.5 | 9.7 | 3.3 | 21 | 7.3 | Gln\Lys | Sample | Total mean μmol/L | Within-lab | | Between-lab | | Reproducibility | | | --- | --- | --- | --- | --- | --- | --- | --- | | | | SD | CV% | SD | CV% | SD | CV% | | 1 | 517 | 35 | 6.9 | 10 | 2.1 | 37 | 7.2 | | 2 | 738 | 45 | 6.2 | 12 | 1.6 | 47 | 6.4 | | 3 | 1409 | 110 | 7.8 | 52 | 3.7 | 121 | 8.6 | 15 {15} Gly | Sample | Total mean μmol/L | Within-lab | | Between-lab | | Reproducibility | | | --- | --- | --- | --- | --- | --- | --- | --- | | | | SD | CV% | SD | CV% | SD | CV% | | 1 | 320 | 23 | 7.5 | 12 | 3.8 | 26 | 8.4 | | 2 | 592 | 37 | 6.3 | 25 | 4.2 | 44 | 7.6 | | 3 | 1398 | 95 | 6.8 | 62 | 4.5 | 114 | 8.1 | Leu\Ile\Pro-OH | Sample | Total mean μmol/L | Within-lab | | Between-lab | | Reproducibility | | | --- | --- | --- | --- | --- | --- | --- | --- | | | | SD | CV% | SD | CV% | SD | CV% | | 1 | 179 | 9.2 | 5.2 | 5.2 | 2.9 | 10 | 5.9 | | 2 | 273 | 12 | 4.5 | 8.0 | 2.9 | 14 | 5.3 | | 3 | 549 | 30 | 5.5 | 22 | 4.0 | 37 | 6.8 | Met | Sample | Total mean μmol/L | Within-lab | | Between-lab | | Reproducibility | | | --- | --- | --- | --- | --- | --- | --- | --- | | | | SD | CV% | SD | CV% | SD | CV% | | 1 | 15 | 1.4 | 9.2 | 0.37 | 2.4 | 1.5 | 9.5 | | 2 | 96 | 5.7 | 5.9 | 5.4 | 5.6 | 7.8 | 8.2 | | 3 | 337 | 21 | 6.3 | 23 | 7.1 | 31 | 9.5 | Orn | Sample | Total mean μmol/L | Within-lab | | Between-lab | | Reproducibility | | | --- | --- | --- | --- | --- | --- | --- | --- | | | | SD | CV% | SD | CV% | SD | CV% | | 1 | 112 | 8.8 | 7.9 | 9.2 | 8.2 | 12 | 11 | | 2 | 200 | 8.4 | 4.2 | 1.3 | 0.62 | 8.5 | 4.2 | | 3 | 484 | 23 | 4.8 | 3.7 | 0.76 | 23 | 4.9 | Phe | Sample | Total mean μmol/L | Within-lab | | Between-lab | | Reproducibility | | | --- | --- | --- | --- | --- | --- | --- | --- | | | | SD | CV% | SD | CV% | SD | CV% | | 1 | 62 | 3.1 | 5.0 | 0.48 | 0.77 | 3.1 | 5.0 | | 2 | 190 | 9.2 | 4.8 | 2.5 | 1.3 | 9.5 | 5.0 | | 3 | 573 | 31 | 5.5 | 10 | 1.9 | 33 | 5.8 | 16 {16} Pro | Sample | Total mean μmol/L | Within-lab | | Between-lab | | Reproducibility | | | --- | --- | --- | --- | --- | --- | --- | --- | | | | SD | CV% | SD | CV% | SD | CV% | | 1 | 150 | 8.8 | 5.8 | 4.0 | 2.6 | 9.6 | 6.4 | | 2 | 304 | 13 | 4.5 | 4.3 | 1.4 | 14 | 4.7 | | 3 | 759 | 37 | 5.0 | 24 | 3.2 | 45 | 5.9 | Tyr | Sample | Total mean μmol/L | Within-lab | | Between-lab | | Reproducibility | | | --- | --- | --- | --- | --- | --- | --- | --- | | | | SD | CV% | SD | CV% | SD | CV% | | 1 | 57 | 3.9 | 6.8 | 1.1 | 1.9 | 4.0 | 7.0 | | 2 | 227 | 11 | 5.0 | 8.3 | 3.6 | 13 | 6.2 | | 3 | 736 | 41 | 5.6 | 26 | 3.6 | 49 | 6.7 | Val | Sample | Total mean μmol/L | Within-lab | | Between-lab | | Reproducibility | | | --- | --- | --- | --- | --- | --- | --- | --- | | | | SD | CV% | SD | CV% | SD | CV% | | 1 | 191 | 9.9 | 5.2 | 4.4 | 2.3 | 10 | 5.7 | | 2 | 295 | 12 | 4.2 | 8.3 | 2.8 | 15 | 5.1 | | 3 | 601 | 33 | 5.5 | 12 | 2.1 | 35 | 5.9 | C0 | Sample | Total mean μmol/L | Within-lab | | Between-lab | | Reproducibility | | | --- | --- | --- | --- | --- | --- | --- | --- | | | | SD | CV% | SD | CV% | SD | CV% | | 1 | 22 | 1.3 | 6.0 | 1.1 | 4.9 | 1.7 | 7.8 | | 2 | 75 | 3.4 | 4.5 | 3.4 | 4.4 | 4.8 | 6.3 | | 3 | 233 | 13 | 5.8 | 6.8 | 2.9 | 15 | 6.5 | C2 | Sample | Total mean μmol/L | Within-lab | | Between-lab | | Reproducibility | | | --- | --- | --- | --- | --- | --- | --- | --- | | | | SD | CV% | SD | CV% | SD | CV% | | 1 | 11 | 0.63 | 5.5 | 0.38 | 3.4 | 0.74 | 6.4 | | 2 | 20 | 0.98 | 4.8 | 0.55 | 2.7 | 1.1 | 5.5 | | 3 | 46 | 2.6 | 5.6 | 1.8 | 3.8 | 3.1 | 6.7 | 17 {17} C3 | Sample | Total mean μmol/L | Within-lab | | Between-lab | | Reproducibility | | | --- | --- | --- | --- | --- | --- | --- | --- | | | | SD | CV% | SD | CV% | SD | CV% | | 1 | 1.2 | 0.07 | 5.9 | 0.04 | 3.5 | 0.08 | 6.8 | | 2 | 8.5 | 0.39 | 4.6 | 0.41 | 4.8 | 0.57 | 6.7 | | 3 | 29 | 1.9 | 6.3 | 1.9 | 6.3 | 2.7 | 8.9 | C4 | Sample | Total mean μmol/L | Within-lab | | Between-lab | | Reproducibility | | | --- | --- | --- | --- | --- | --- | --- | --- | | | | SD | CV% | SD | CV% | SD | CV% | | 1 | 0.14 | 0.01 | 9.8 | <0.01 | 1.8 | 0.01 | 10. | | 2 | 1.3 | 0.06 | 5.1 | 0.07 | 5.6 | 0.10 | 7.6 | | 3 | 4.6 | 0.30 | 6.5 | 0.28 | 6.1 | 0.41 | 8.9 | C5 | Sample | Total mean μmol/L | Within-lab | | Between-lab | | Reproducibility | | | --- | --- | --- | --- | --- | --- | --- | --- | | | | SD | CV% | SD | CV% | SD | CV% | | 1 | 0.10 | 0.02 | 18 | <0.01 | 8.8 | 0.02 | 20 | | 2 | 2.6 | 0.13 | 4.8 | 0.09 | 3.3 | 0.15 | 5.8 | | 3 | 10 | 0.56 | 5.5 | 0.45 | 4.4 | 0.71 | 7.0 | C5DC\C6OH | Sample | Total mean μmol/L | Within-lab | | Between-lab | | Reproducibility | | | --- | --- | --- | --- | --- | --- | --- | --- | | | | SD | CV% | SD | CV% | SD | CV% | | 1 | 0.058 | 0.01 | 21 | <0.01 | 8.6 | 0.01 | 22 | | 2 | 1.0 | 0.07 | 6.8 | 0.06 | 5.8 | 0.09 | 9.0 | | 3 | 3.8 | 0.30 | 7.7 | 0.27 | 7.1 | 0.40 | 10 | C6 | Sample | Total mean μmol/L | Within-lab | | Between-lab | | Reproducibility | | | --- | --- | --- | --- | --- | --- | --- | --- | | | | SD | CV% | SD | CV% | SD | CV% | | 1 | 0.041 | <0.01 | 19 | <0.01 | 7.5 | <0.01 | 20 | | 2 | 0.96 | 0.05 | 4.9 | 0.04 | 4.3 | 0.06 | 6.5 | | 3 | 3.7 | 0.22 | 6.0 | 0.21 | 5.6 | 0.30 | 8.2 | 18 {18} C8 | Sample | Total mean μmol/L | Within-lab | | Between-lab | | Reproducibility | | | --- | --- | --- | --- | --- | --- | --- | --- | | | | SD | CV% | SD | CV% | SD | CV% | | 1 | 0.07 | 0.01 | 16 | <0.01 | 13 | 0.02 | 20 | | 2 | 1.9 | 0.11 | 5.7 | 0.07 | 4.0 | 0.13 | 6.9 | | 3 | 7.2 | 0.43 | 6.0 | 0.35 | 4.8 | 0.55 | 7.6 | C10 | Sample | Total mean μmol/L | Within-lab | | Between-lab | | Reproducibility | | | --- | --- | --- | --- | --- | --- | --- | --- | | | | SD | CV% | SD | CV% | SD | CV% | | 1 | 0.08 | <0.01 | 8.3 | <0.01 | 3.8 | <0.01 | 9.1 | | 2 | 0.87 | 0.04 | 4.9 | 0.05 | 5.9 | 0.07 | 7.7 | | 3 | 3.2 | 0.21 | 6.6 | 0.22 | 6.8 | 0.30 | 9.5 | C12 | Sample | Total mean μmol/L | Within-lab | | Between-lab | | Reproducibility | | | --- | --- | --- | --- | --- | --- | --- | --- | | | | SD | CV% | SD | CV% | SD | CV% | | 1 | 0.04 | <0.01 | 15 | <0.01 | 9.0 | <0.01 | 17 | | 2 | 0.77 | 0.05 | 6.2 | 0.02 | 2.8 | 0.05 | 6.8 | | 3 | 2.9 | 0.19 | 6.4 | 0.10 | 3.4 | 0.21 | 7.2 | C14 | Sample | Total mean μmol/L | Within-lab | | Between-lab | | Reproducibility | | | --- | --- | --- | --- | --- | --- | --- | --- | | | | SD | CV% | SD | CV% | SD | CV% | | 1 | 0.11 | <0.01 | 8.8 | <0.01 | 4.8 | 0.01 | 10 | | 2 | 1.1 | 0.06 | 5.4 | 0.06 | 5.6 | 0.09 | 7.8 | | 3 | 4.0 | 0.23 | 5.8 | 0.28 | 7.0 | 0.37 | 9.1 | C16 | Sample | Total mean μmol/L | Within-lab | | Between-lab | | Reproducibility | | | --- | --- | --- | --- | --- | --- | --- | --- | | | | SD | CV% | SD | CV% | SD | CV% | | 1 | 1.1 | 0.07 | 6.0 | 0.06 | 5.0 | 0.09 | 7.8 | | 2 | 8.1 | 0.37 | 4.6 | 0.31 | 3.9 | 0.49 | 6.0 | | 3 | 27 | 1.4 | 5.2 | 1.3 | 4.6 | 1.9 | 6.9 | 19 {19} # **C18** | Sample | Total mean μmol/L | Within-lab | | Between-lab | | Reproducibility | | | --- | --- | --- | --- | --- | --- | --- | --- | | | | SD | CV% | SD | CV% | SD | CV% | | 1 | 0.65 | 0.03 | 5.3 | 0.01 | 1.6 | 0.04 | 5.5 | | 2 | 2.1 | 0.11 | 5.2 | 0.04 | 1.7 | 0.12 | 5.5 | | 3 | 6.5 | 0.38 | 5.8 | 0.18 | 2.7 | 0.42 | 6.4 | # **SA** | Sample | Total mean μmol/L | Within-lab | | Between-lab | | Reproducibility | | | --- | --- | --- | --- | --- | --- | --- | --- | | | | SD | CV% | SD | CV% | SD | CV% | | 1 | 0.28 | 0.08 | 26 | 0.05 | 17 | 0.09 | 32 | | 2 | 7.0 | 0.72 | 10 | 0.76 | 10 | 1.0 | 14 | | 3 | 28 | 2.4 | 8.4 | 3.1 | 10 | 3.9 | 13 | # **ADO** | Sample | Total mean μmol/L | Within-lab | | Between-lab | | Reproducibility | | | --- | --- | --- | --- | --- | --- | --- | --- | | | | SD | CV% | SD | CV% | SD | CV% | | 1 | 0.37 | 0.04 | 10 | 0.02 | 5.4 | 0.04 | 12 | | 2 | 2.0 | 0.14 | 6.8 | 0.11 | 5.2 | 0.17 | 8.6 | | 3 | 7.9 | 0.44 | 5.5 | 0.28 | 3.6 | 0.52 | 6.5 | # **C26:0-LPC** | Sample | Total mean μmol/L | Within-lab | | Between-lab | | Reproducibility | | | --- | --- | --- | --- | --- | --- | --- | --- | | | | SD | CV% | SD | CV% | SD | CV% | | 1 | 0.22 | 0.04 | 16 | 0.04 | 17 | 0.05 | 24 | | 2 | 0.58 | 0.07 | 11 | 0.06 | 10 | 0.09 | 15 | | 3 | 1.8 | 0.15 | 8.3 | 0.20 | 11 | 0.25 | 13 | # *b. Linearity/assay reportable range:* Linearity on the NeoBase 2 was determined by testing dried blood spots with 12 analyte levels spanning the ranges defined below in three different linearity studies per analyte. A spike and recovery study was also conducted. Summary of these studies are presented in the tables below. 20 {20} | Analyte | Linear range lower limit (μmol/L) | Linear range upper limit (μmol/L) | | --- | --- | --- | | Ala | 145 | 1580 | | Arg | 1.95 | 311 | | Asa | 0.42 | 67.7 | | Cit | 10.1 | 934 | | Gln\Lys | 38.7 | 2190 | | Gly | 264 | 1820 | | Leu\Ile\Pro-OH | 56.6 | 1420 | | Met | 2.37 | 826 | | Orn | 30.1 | 799 | | Phe | 21.9 | 1470 | | Pro | 39.4 | 1250 | | Tyr | 19.8 | 1240 | | Val | 55.8 | 1110 | | C0 | 8.16 | 392 | | C2 | 3.52 | 139 | | C3 | 0.44 | 58.4 | | C4 | 0.06 | 10.4 | | C5 | 0.05 | 17.0 | | C5DC\C6OH | 0.03 | 6.99 | | C6 | 0.02 | 7.48 | | C8 | 0.06 | 38.7 | | C10 | 0.03 | 6.93 | | C12 | 0.02 | 9.15 | | C14 | 0.05 | 8.61 | 21 {21} | C16 | 1.00 | 51.5 | | --- | --- | --- | | C18 | 0.48 | 11.8 | | SA | 0.28 | 122 | | ADO | 0.18 | 36.9 | | C26:0-LPC | 0.22 | 7.46 | Results from the three replicate linearity runs: | Analyte | R² Range | Slope Range | | --- | --- | --- | | Ala | 0.994 - 0.997 | 0.696 - 0.786 | | Arg | 0.992 - 0.995 | 0.655 - 0.799 | | Asa-Total | 0.992 - 0.993 | 0.193 - 0.219 | | Cit | 0.995 - 0.997 | 0.681 - 0.786 | | Gln | 0.994 - 0.996 | 0.719 - 0.830 | | Gly | 0.995 - 0.998 | 0.697 - 0.819 | | Leu\Ile\Pro-OH | 0.996 - 0.998 | 0.680 - 0.762 | | Met | 0.998 - 0.998 | 0.665 - 0.741 | | Orn | 0.998 - 0.998 | 0.612 - 0.673 | | Phe | 0.996 - 0.998 | 0.678 - 0.796 | | Pro | 0.996 - 0.999 | 0.648 - 0.708 | | Tyr | 0.997 - 0.999 | 0.692 - 0.826 | | Val | 0.995 - 0.998 | 0.701 - 0.789 | | C0 | 0.996 - 0.998 | 0.764 - 0.858 | | C2 | 0.996 - 0.998 | 0.725 - 0.835 | | C3 | 0.996 - 0.998 | 0.870 - 1.047 | | C4 | 0.997 - 0.997 | 0.699 - 0.802 | | C5 | 0.997 - 0.999 | 0.635 - 0.723 | | C5DC\C6OH | 0.998 - 0.998 | 0.587 - 0.685 | | C6 | 0.998 - 0.998 | 0.702 - 0.812 | | C8 | 0.996 - 0.998 | 0.705 - 0.818 | | C10 | 0.997 - 0.998 | 0.664 - 0.803 | | C12 | 0.997 - 0.998 | 0.743 - 0.836 | | C14 | 0.997 - 0.999 | 0.766 - 0.888 | | C16 | 0.994 - 0.996 | 0.855 - 0.992 | | C18 | 0.997 - 0.998 | 0.823 - 0.924 | | C26 | 0.999 - 0.999 | 1.247 - 1.768 | | SA | 0.998 - 0.999 | 0.499 - 0.519 | | ADO | 0.999 - 0.999 | 0.958 - 1.098 | | C26:0-LPC | 0.996 - 0.998 | 1.228 - 1.320 | 22 {22} ### Recovery: Analyte recovery for the NeoBase Non-derivatized MSMS kit was evaluated using the Perkin Elmer TQD tandem mass spectrometry system over a total of 21 runs, consisting of samples with 5 different levels analyzed in 6 replicates per run. The results are summarized below: | Analyte | Recovery with kit lot 1 (%) | Recovery with kit lot 2 (%) | Recovery with kit lot 3 (%) | Mean recovery over kit lots (%) | | --- | --- | --- | --- | --- | | ADO | 101.8 | 89.3 | 98.9 | 96.7 | | Ala | 103.3 | 107.1 | 111.2 | 107.2 | | Arg | 87.2 | 75.4 | 84.8 | 82.5 | | Asa | 27.5 | 23.1 | 25.9 | 25.5 | | Cit | 92.6 | 82.1 | 89.8 | 88.1 | | Gln | 107.9 | 102.1 | 109.7 | 106.5 | | Gly | 105.7 | 87.9 | 94.5 | 96.0 | | Leu | 95.3 | 85.0 | 91.7 | 90.7 | | Met | 89.1 | 78.8 | 85.2 | 84.4 | | Orn | 84.5 | 78.0 | 84.2 | 82.2 | | Phe | 94.7 | 81.3 | 88.0 | 88.0 | | Pro | 95.3 | 81.0 | 88.1 | 88.1 | | SA | 48.9 | 46.0 | 50.1 | 48.4 | | Tyr | 92.0 | 82.2 | 88.7 | 87.6 | | Val | 103.6 | 90.2 | 98.0 | 97.3 | | C0 | 123.9 | 97.9 | 107.2 | 109.7 | | C2 | 107.7 | 96.6 | 102.3 | 102.2 | | C3 | 125.4 | 109.1 | 119.3 | 118.0 | | C4 | 91.9 | 78.6 | 86.6 | 85.7 | | C5 | 87.0 | 74.6 | 82.2 | 81.3 | | C5DC | 82.5 | 72.9 | 79.4 | 78.2 | | C6 | 99.6 | 84.6 | 95.4 | 93.2 | | C8 | 99.1 | 86.1 | 95.1 | 93.4 | | C10 | 103.0 | 89.9 | 99.0 | 97.3 | | C12 | 94.1 | 83.1 | 94.1 | 90.4 | | C14 | 97.6 | 86.5 | 95.0 | 93.0 | | C16 | 120.7 | 107.4 | 117.6 | 115.3 | | C18 | 105.5 | 96.7 | 103.8 | 102.0 | | C26 | 122.2 | 109.6 | 123.8 | 118.5 | | C26:0-LPC | 109.2 | 110.0 | 118.3 | 112.5 | The following limitation is included in the package insert for Asa: “Please note that the 25.5% recovery of Asa (slope range =0.193-0.219) is lower compared to other analytes measured with NeoBase 2 Non-derivatized MSMS assay (Table 12). Incomplete recovery may cause difference in measured analyte level 23 {23} compared to alternative methods. Therefore, it is important that each laboratory establishes its own reference range and cut-off values with the NeoBase 2 Non-derivatized MSMS assay." c. Traceability, Stability, Expected values (controls, calibrators, or methods): NeoBase 2 Non-derivatized MSMS kit is traceable to NIST standards and has been anchored to the non-labeled reference compounds, which are the primary calibrators of the kit. The concentration of labelled secondary calibrators is assigned against the primary calibrators. d. Detection limit: 15 serially diluted dried blood spots samples in a blood matrix spiked with internal standards were used for the analytical sensitivity study. Extracted samples were pipetted onto the plates in 6 replicates of each, from the lowest dilution level to the highest dilution level. At the below listed concentrations the sponsor claims that they meet an imprecision criteria of CV less than or equal to 20%. | Analyte | Analytical sensitivity limit (μmol/L) | | --- | --- | | Ala | 1.75 | | Arg | 0.37 | | \( Asa^1 \) | 0.18 | | Cit | 2.64 | | Gln\Lys | 0.81 | | Gly | 4.35 | | Leu\Ile\Pro-OH | 0.40 | | Met | 6.31 | | Orn | 1.75 | | Phe | 0.29 | | Pro | 0.35 | | Tyr | 1.75 | | Val | 0.22 | | C0 | 0.17 | | C2 | 0.04 | | C3 | 0.02 | | C4 | 0.01 | | C5 | 0.02 | | C5DC\C6OH | 0.02 | 24 {24} | Analyte | Analytical sensitivity limit (μmol/L) | | --- | --- | | C6 | 0.05 | | C8 | 0.01 | | C10 | 0.01 | | C12 | 0.01 | | C14 | 0.01 | | C16 | 0.02 | | C18 | 0.01 | | SA | 0.24 | | ADO | 0.08 | | C26:0-LPC | 0.08 | ### e. Analytical specificity: Dried blood spot samples representing two different analyte concentrations were tested in an interference study. The substances tested for interference included endogenous substances and possible sample contaminants. The effect of 25 substances was assessed by the paired-difference method at two analyte concentration levels (both normal and abnormal) with the NeoBase 2 Non-derivatized MSMS kit. Additionally, 9 substances that are abundant in the DBS sample matrix and have potential for unspecific effects on the test results were studied. Results confirmed that 11 of the 25 substances caused a significant change in the test result. These 11 compounds were further evaluated by dose-response testing. At the following concentrations, the bias between the test and control samples did not exceed 15% with overall 95% confidence. The sponsor claims that the following substances were found not to interfere with the assay at the concentration indicated. | Tested substance | Added concentration of tested substance in blood | | --- | --- | | Formiminoglutamic acid (Figlu) | 37.1 μmol/L | | O-Acetyl-L-serine | 1000 μmol/L | | 6-Aminocaproic Acid | 6.07 μmol/L | | DL-Malic acid | 3000 μmol/L | | 4-Aminoantipyrine | 500 μmol/L | | Propranolol | 7.74 μmol/L | | 2,5-dihydroxybenzoic acid | 127 μmol/L | | Bilirubin conjugated | 15 mg/dL | | Bilirubin unconjugated | 10 mg/dL | | Calcifediol | 250 nmol/L | | Acetaminophen | 5.5 mg/dL | 25 {25} For the substances that did interfere with the assay, the following was added to the package insert of the device: Sarcosine: Amino acid derivative Sarcosine was found to interfere with the assay by increasing the measured Ala concentration. Sarcosine concentrations above 62.5 μmol/L may cause a false positive screening result. Sarcosine concentration in plasma ranges from 0–625 μmol/L in newborns aged 0–1 months. However, Sarcosine does not exist in healthy newborns; it is only found at detectable amount when a newborn is affected by hypersarcosinemia. Therefore, Sarcosine is unlikely to interfere with Ala in routine testing. Creatine: Non-essential amino acid Creatine was found to interfere with the assay by increasing the measured Ala and Leu concentrations. Creatine concentrations above 1500 μmol/L with Ala or above 4500 μmol/L with Leu may cause a false positive screening result. The normal expected Creatine level in newborns aged 0–1 months is 107–640 μmol/L. Therefore, Creatine is unlikely to interfere with Ala and Leu in routine testing. Verapamil metabolite D617: D617 is a metabolite of calcium channel blocker Verapamil. D617 was found to interfere with the assay by increasing the measured Asa concentration. D617 concentrations from 0.97 μmol/L may cause a false positive screening result on Asa. Therapeutic concentration range in plasma for Verapamil is 0.11–1.32 μmol/L. D617 has been found to present appr. 20% of the given oral dose excreted in to urine, i.e. the D617 level is very unlikely to exceed the concentration of 0.97 μmol/L in whole blood. Therefore, Verapamil metabolite D617 is unlikely to interfere with Asa in routine testing. Nevertheless, newborns given Verapamil or exposed to the compound during pregnancy or breastfeeding could screen positive for Asa. L-Lysine: L-Lysine was found to interfere with the assay by increasing the measured Gln and Glu concentrations. L-Lysine is an essential amino acid and is isobaric to NeoBase 2 analyte Gln. NeoBase 2 assay cannot separate the compounds, and the result of Gln is a sum of Gln and L-Lysine (Gln\Lys). The reference plasma level of L-Lysine, Gln and Glu in newborns aged 0–1 months are 92–325 μmol/L, 376–709 μmol/L and 62–620 μmol/L, respectively. L-Lysine may cause a false positive screening result to Gln. The proposed cut-off area measured with NeoBase 2 assay for Gln is generally high (e.g. 99.0% percentile, 1120 μmol/L). An additional 730 μmol/L dose of L-Lysine would be needed to raise the Gln concentration from endogenous level (465 μmol/L) to the cut-off area (1100 μmol/L). For Glu, L-Lysine concentrations from 355 μmol/L may cause a false positive screening result. Since the whole blood used in the interference samples contains also endogenous L-Lysine, the sum of spiked and endogenous L-Lysine levels are in the upper part of newborn physiological L-Lysine range. In hyperlysinemia, the concentration of L-Lysine in blood plasma is relatively high. Plasma L-Lysine levels have been reported to exceed 600 μmol/L and can reach up to 2000 μmol/L. When blood specimen is taken from a 26 {26} newborn with such a condition, L-Lysine may cause false positive screening results to Gln and/or Glu. L-Glutamic acid: Non-essential amino acid and NeoBase 2 analyte L-Glutamic acid (Glu) was found to interfere with the assay by increasing the measured Met concentration. Glu concentrations above 2250 μmol/L may cause a false positive screening result on Met. The reference plasma level of Glu in newborns aged 0–1 months is 62–620 μmol/L. Therefore, Glu is very unlikely to interfere with Met in routine testing. L-Asparagine: Non-essential amino acid L-Asparagine was found to interfere with the assay by increasing the measured Orn concentration. L-Asparagine concentrations above 375 μmol/L may cause a false positive screening result on Orn. The reference plasma level of L-Asparagine in newborns aged 0–1 months is 29–132 μmol/L. Therefore L-Asparagine is unlikely to interfere with Orn in routine testing. L-Ornithine (Orn): NeoBase 2 analyte L-Ornithine (Orn) was found to interfere with the assay by increasing the measured Pro concentration. The interference is caused by mass transition overlap between a fragment of Orn formed in the ion source and Pro. Orn concentrations above 188 μmol/L may cause a false positive screening result on Pro. The proposed cut-off area measured with NeoBase 2 assay for Pro is above 200 μmol/L (e.g. 99.0% percentile, 235 μmol/L). An additional 638 μmol/L dose of Orn would be needed to raise the Pro concentration from endogenous level (155 μmol/L) to the cut-off area (240 μmol/L). The proposed normal population for Orn measured with NeoBase 2 assay (e.g. 99% percentiles range between 152–194 μmol/L) is on much lower concentration level than the needed additional dose. It is unlike that Orn interferes with Pro in routine testing. L-Methionine sulfone: Amino acid derivative L-Methionine sulfone was found to interfere with the assay by increasing the measured Tyr concentration. L-Methionine sulfone concentrations above 15.6 μmol/L may cause a false positive screening result on Tyr. No reference concentration in newborns was found for methionine sulfone. However, because methionine sulfone is an oxidation product of methionine sulfoxide, and methionine sulfoxide is a product of methionine (Met), the highest concentration of methionine sulfone should not exceed the normal level of Met in infants aged 0–1 year (10–60 μmol/L). L-Methionine sulfone dose of 62.5 μmol/L increased the endogenous Tyr concentration (53 μmol/L) to 76 μmol/L. Since the proposed cut-off area measured with NeoBase 2 assay for Tyr is higher (e.g. 99.0% percentile, 188 μmol/L), L-Methionine sulfone is unlikely to interfere routine testing. Lidocaine: Lidocaine was found to interfere with the assay by decreasing the measured C4 concentration. Lidocaine concentrations above 25.6 μmol/L may cause a false negative screening result on C4. Lidocaine is a drug used to e.g. numb tissues. If disinfectant pads containing lidocaine are used to wipe off the heels of newborns in specimen collection, there is potential for lidocaine to be carried into the specimen. However, the residual amount of this compound present after skin preparation should 27 {27} only be a fraction of the effective interfering level and the less likely to interfere. Therapeutic concentration range in plasma for Lidocaine is 5.1–25.6 μmol/L. Concentrations above 25.6 μmol/L are reported as toxic. Therefore, Lidocaine is unlikely to interfere routine testing. Albumin: High albumin concentrations were found to interfere with the assay. When total albumin is above 3.16 g/dL, the interference caused an increase in the measured ADO concentrations. The reference range for albumin in infant plasma/serum aged 0–12 months is 2.8–4.7 g/dL corresponding to albumin concentration of 1.4–2.4 g/dL in whole blood. Intralipid (Triglyceride): High intralipid concentrations were found to interfere with the assay. When total triglycerides are above 0.82 g/dL, the interference caused an increase in the measured Glu concentration. When more than 0.10 g/dL of intralipid was added to blood containing 0.07 g/dL of endogenous triglycerides (i.e. tested total triglycerides above 0.17 g/dL) the interference caused an increase in the measured C24:0-LPC concentration. The reference range for triglycerides in newborns aged 0–7 days has been reported to be from 0.02 to 0.18 g/dL in serum corresponding to triglyceride concentration in whole blood of 0.01 to 0.09 g/dL. Chlorhexidine digluconate: Chlorhexidine digluconate was found to interfere with the assay by increasing the measured C24:0-LPC and C26:0-LPC concentrations. Chlorhexidine digluconate is a cationic broad-spectrum antimicrobial agent belonging to the bis(biguanide) family. Its mechanism of action involves destabilization of the outer bacterial membrane. Chlorhexidine digluconate amounts above 0.03% may cause false positive screening results on C24:0-LPC and C26:0-LPC. If disinfectant pads containing chlorhexidine digluconate are used to wipe off the heels of newborns in preparation for sample collection, there is potential for chlorhexidine digluconate to be carried into the sample. It can be estimated that in the worst case, 1 μL of the 3% skin disinfection solution might contaminate a 75 μL blood droplet, which corresponds to an amount of 0.04% Chlorhexidine digluconate in the sample. Therefore, it is unlikely that Chlorhexidine digluconate will interfere with C24:0-LPC and C26:0-LPC in routine testing. Hemoglobin: High hemoglobin concentrations were found to interfere with the assay. Total hemoglobin above 21.7 g/dL caused a decrease in the measured C24:0-LPC by 19% and in the measured C26:0-LPC by 28%. Total hemoglobin above 20.4 g/dL caused an elevation in the measured ADO by 20%. The effect of hematocrit on Arg was evaluated. Hematocrit values below 43% increased Arg results by 20%, and hematocrit values above 63% decreased Arg results by 30%. Although interference by hemoglobin and hematocrit was observed with concentrations within the newborn reference ranges (12.0–22.0 g/dL for hemoglobin, 35–65% for hematocrit), it is concluded based on the external study results that the interferences are not pronounced enough to impair the separation of the affected and un-affected cases. Interference by hemoglobin could cause false 28 {28} negative screening results only if C24:0-LPC would be used as the sole marker for X-ALD. C24:0-LPC should always be used together with the primary marker C26:0-LPC. Benzocaine: Disinfectants such as alcohol swabs with a topical anesthetic benzocaine should not be used to wipe off the heel of a newborn. Benzocaine and Phe are isomers having the same mass to charge ratio of 166.1. Therefore, benzocaine may cause falsely elevated Phe concentration and a false positive phenylketonuria (PKU) screening result. C5 isomer pivalylcarnitine: Pivalic acid may cause false positive screening result for Isovaleric acidemia (IVA), whose marker is acylcarnitine C5. Pivalic acid can be liberated from a prodrug containing esterified pivalic acid (such as pivalic-ester containing antibiotics, corticosteroids or other pharmaceuticals) administered to mothers or newborns. Pivalic acid is further metabolized to pivalylcarnitine which is an isomer of C5, and therefore pivalic acid can cause falsely elevated C5 results. Falsely elevated C5 concentrations have been reported in newborns due to pivalylcarnitine interference. Administration of pivalic acid containing prodrugs can lead to carnitine depletion. In addition, falsely elevated C5 concentrations have been connected to cases where pivalylcarnitine originated from neopentanoate esters present in nipple-fissure unguent used by the breastfeeding mothers. C8 isomer valproylcarnitine: Medication valproic acid administered to mothers or newborns may interfere with the screening of Medium-chain acyl-CoA dehydrogenase deficiency (MCAD) or Medium-chain ketoacyl-CoA thiolase deficiency (MCKAT), whose diagnostic marker is acylcarnitine C8. Valproic acid is metabolized to valproylcarnitine, which is isomer of C8, and can cause falsely elevated C8 concentration. False positive MCAD results have been measured in newborns due to valproylcarnitine interference. C3DC\C4OH, C4DC\C5OH and C5DC\C6OH: Analytes in the pairs C3DC\C4OH; C4DC\C5OH; and C5DC\C6OH; are all natural acylcarnitines that can be present in dried blood spots and cannot be separated in the NeoBase 2 assay due to mass transition overlap. The mass to charge ratios of the precursor ions are 248 (for C3DC, C4OH), 262 (for C4DC, C5OH), and 276 (for C5DC, C6OH) and they all have the same identifying product ion (m/z 85). As a result, the NeoBase 2 Non-derivatized MSMS kit reports the results for these analytes in the pair together as a sum, which is very much the same as the case for Leu\Ile\Hydroxyproline, and Gln\Lys. Because of this overlap, the results for these analytes should be reported as the cumulative concentration of the two analytes in the pair. C16:1OH\C17: C16:1OH is a marker among other hydroxylated long chain acylcarnitines for Long-chain L-3-hydroxyacyl-CoA dehydrogenase deficiency (LCHAD) and Trifunctional protein deficiency (TFP). Heptadecanoylcarnitine (C17) has been identified as a marker specific for Propionic acidemia (PROP) and Methylmalonic acidemia (MUT). C17 and C16:1OH are isomers, having the same 29 {29} mass to charge ratio of m/z 414, and are always measured in NeoBase 2 assay as a sum of both analytes. In screening of LCHAD, TFP, PROP and MUT, the cumulative sum concentration of C16:1OH and C17 increases. It is recommended to confirm positive screening result with 2nd tier analysis, which is capable of separating C16:1OH and C17 and identify specifically the disorder. C26:0-LPC: Elevated C26:0-LPC concentrations have been measured in newborn blood spots and post-natal blood specimens taken from children diagnosed by Aicardi Goutières Syndrome (AGS) leading to false positive results in first tier X-ALD screening. Interference from M+2 Isotopic Peaks: The analytes and internal standards measured in NeoBase 2 assay not only produce an M peak (the monoisotopic peak that is used in the measurement), but also M+1, M+2, and M+3 peaks. These additional M+n peaks are due to the naturally occurring heavier stable isotopes such as 13C, 15N or 18O. In tandem mass spectra of complex samples where many analytes are analyzed simultaneously (as is the case of the NeoBase 2 assay) the M+n peaks of one compound have the potential to overlap with the peaks generated by other compounds of neighbouring m/z and cause falsely elevated peaks. Potential M+2 peak interferences are as follows: M+2 peak of C5 overlaps with C3DC\C4OH; M+2 peak of C6 overlaps with C4DC\C5OH; and M+2 peak of C8 overlaps with C6DC. However, the effect is only significant when C5, C6, and C8 are present in high concentrations. At the endogenous concentrations the risk for false positive result with C3DC\C4OH, C4DC\C5OH and C6DC is negligible. When elevated level of C5, which is a marker for Isovaleric acidemia (IVA) and 2-Methylbutyrylglycinuria (2MBG), is observed, concentration of C3DC\C4OH must be evaluated. When elevated level of C6, which is a marker for Medium-chain acyl-CoA dehydrogenase deficiency (MCAD), is observed, concentration of C4DC\C5OH must be evaluated. When elevated level of C8, which is a marker for MCAD and Medium-chain ketoacyl-CoA thiolase deficiency (MCKAT), is observed, concentration of C6DC must be evaluated. Conversely, when elevations are detected on C3DC\C4OH; C4DC\C5OH; or C6DC, it is recommended to evaluate the concentrations of C5, C6, and C8 to ensure these observations are not due to the M+2 effect described here. Plasticizers: Plasticizers or other additives may leach from the plastic material used in the sample preparation, storage packages and medical equipment and interfere with the newborn screening results. For example, slip agent Oleamide (m/z 282) is known to interfere with C5DC IS having the same mass to charge ratio. Elevated C5DC IS intensity falsely decreases acylcarnitine C5DC analyte concentration and may cause false negative screening result on C5DC, which is a marker for Glutaric acidemia type I (GAI). Similarly, a common anti-static agent Lauric acid diethanolamide (LDEA, m/z 288) has been found to interfere with acylcarnitine C8 having the same mass to charge ratio. LDEA can lead to false positive C8 result, which is a marker for disorders Medium-chain acyl-CoA dehydrogenase deficiency (MCAD) and Medium-chain ketoacyl-CoA thiolase deficiency (MCKAT) disorder. In addition, falsely elevated C8 levels in two neonates treated with extracorporeal membrane 30 {30} oxygenation (ECMO) has been identified. The C8 interference was traced to PVC tubing used in ECMO and a plasticizer Di-ethylhexyl phthalate (DEHP) used commonly in the manufacturing of PVC. Interference originated from a DEHP metabolite, 2-Ethylhexanoic acid, which was further metabolized in the exposed neonates to a C8 isomer, 2-ethylhexacosanoylcarnitine, can lead to false positive C8 test result. In the other neonate sample, also falsely elevated acylcarnitine C6DC concentration was detected. Interference was likely because of another plasticizer, di-(2-ethylhexyl) adipate (DEHA) metabolite adipic acid, which was metabolized to C6DC. If DEHA is metabolized to C6DC, which is a marker for 3-Hydroxy-3-methylglutaric acidemia (HMG), the false positive C6DC test result may occur. The NeoBase 2 assay is validated with the specific microplates and plate covers provided with the kit and any other items should not be used to avoid plasticizer or other additive contamination. # Carryover: The carry-over evaluation for the NeoBase2 Non-derivatized MSMS kit was conducted by analyzing two plates using two TQD MSMS Screening Systems. Three low sample replicates were measured immediately after having measured a high sample replicate and the sample sequence was repeated twelve times during one run. No functionally significant carry-over effect was observed with any of the analytes. # Drift: The drift study was performed using several sample types at different concentration levels to indicate possible drift. Neo Multilevel DBS samples, L1, L3 and L6 were selected to demonstrate the drift at endogenous concentrations, near cut-off concentrations and concentrations representing the upper part of the measuring range, respectively. In addition, the drift of the available control materials, NeoBase2 Controls Low (LC) and High (HC), and publicly available QA controls were included as samples in the study. The drift study for the kit was completed via the analysis of 10 plates using several sample types at different concentration levels (from endogenous area to the upper part of the measuring range) to indicate possible drift. The results demonstrate that there is no drift for 35 hours continuous run time with the TQD, which is the maximum continuous run time when the autosampler is fully loaded. The fully prepared and sealed assay plate can be stored in the MSMS system autosampler at ambient temperature for 31 hours before the assay run starts. # f. Assay cut-off: Not applicable. # 2. Comparison studies: a. Method comparison with predicate device: 31 {31} See clinical performance data below. # b. *Matrix comparison:* Not applicable. This device is for use with newborn dried blood spots only. # 3. Clinical studies: # a. *Clinical Sensitivity:* Not applicable. # b. *Clinical specificity:* Not applicable. # c. Other clinical supportive data (when a. and b. are not applicable): A comparison of results from neonatal dried blood samples from two state laboratories within the United States and known positive neonatal dried blood samples from sample banks using the NeoBase 2 assay on the TQD system and the predicate test system was performed. Testing was performed with a total of 4456 presumptive negative neonatal dried blood samples. Included in the 4456 samples were 76 dried blood samples from affected babies, randomly distributed throughout the study. The overall percent agreement is presented in the tables below at the different cut-offs. # **Agreement between NeoBase2 and NeoBase Amino acids and Ketones with 99.5% cutoff** | Analyte | N | Overall Agreement | | --- | --- | --- | | Ala | 4456 | 99.5 % | | Arg | 4456 | 99.8 % | | Cit | 4456 | 99.3 % | | Gly | 4456 | 99.6 % | | Met | 4456 | 99.7 % | | Orn | 4456 | 99.1 % | | Phe | 4456 | 99.4 % | | Pro | 4456 | 99.7 % | | Tyr | 4456 | 99.7 % | | Val | 4456 | 99.6 % | | SA | 4456 | 99.0 % | 32 {32} # **Agreement between NeoBase2 and NeoBase Carnitines with 99.5% cutoff** | Analyte | N | Overall Agreement | Analyte | N | Overall Agreement | | --- | --- | --- | --- | --- | --- | | **C0** | 4456 | 99.8 % | **C14** | 4456 | 99.8 % | | **C3** | 4456 | 99.9 % | **C14:1** | 4456 | 99.6 % | | **C4** | 4456 | 99.8 % | **C14:2** | 4456 | 99.7 % | | **C5** | 4456 | 99.6 % | **C14OH** | 4456 | 99.4 % | | **C5:1** | 4456 | 97.9 % | **C16** | 4456 | 99.8 % | | **C6** | 4456 | 99.9 % | **C16:1** | 4456 | 99.8 % | | **C6DC** | 4456 | 99.8 % | **C16OH** | 4456 | 99.7 % | | **C8** | 4456 | 99.7 % | **C16:1OH** | 4456 | 99.8 % | | **C8:1** | 4456 | 99.8 % | **C18** | 4456 | 99.5 % | | **C10** | 4456 | 99.8 % | **C18:1** | 4456 | 99.5 % | | **C10:1** | 4456 | 99.8 % | **C18:2** | 4456 | 99.7 % | | **C10:2** | 4456 | 99.7 % | **C18OH** | 4456 | 99.3 % | | **C12** | 4456 | 99.7 % | **C18:1OH** | 4456 | 98.6 % | | **C12:1** | 4456 | 99.8 % | | | | # **Agreement between NeoBase2 and NeoBase Amino acids and Ketones with 99% cutoff** | Analyte | N | Overall Agreement | | --- | --- | --- | | **Ala** | 4456 | 98.5 % | | **Arg** | 4456 | 99.8 % | | **Cit** | 4456 | 98.9 % | | **Gly** | 4456 | 99.1 % | | **Met** | 4456 | 99.7 % | | **Orn** | 4456 | 98.6 % | | **Phe** | 4456 | 99.0 % | | **Pro** | 4456 | 99.0 % | | **Tyr** | 4456 | 99.3 % | | **Val** | 4456 | 99.0 % | | **SA** | 4456 | 98.1 % | # **Agreement between NeoBase2 and NeoBase Carnitines with 99% cutoff** | Analyte | N | Overall Agreement | Analyte | N | Overall Agreement | | --- | --- | --- | --- | --- | --- | | **C0** | 4456 | 99.1 % | **C14** | 4456 | 99.4 % | | **C3** | 4456 | 99.7 % | **C14:1** | 4456 | 99.4 % | | **C4** | 4456 | 99.6 % | **C14:2** | 4456 | 99.6 % | | **C5** | 4456 | 99.3 % | **C14OH** | 4456 | 99.3 % | | **C5:1** | 4456 | 97.9 % | **C16** | 4456 | 99.6 % | | **C6** | 4456 | 99.5 % | **C16:1** | 4456 | 99.6 % | | **C6DC** | 4456 | 99.6 % | **C16OH** | 4456 | 99.4 % | 33 {33} | Analyte | N | Overall Agreement | Analyte | N | Overall Agreement | | --- | --- | --- | --- | --- | --- | | **C8** | 4456 | 99.5 % | **C16:10H** | 4456 | 99.6 % | | **C8:1** | 4456 | 99.6 % | **C18** | 4456 | 99.0 % | | **C10** | 4456 | 99.7 % | **C18:1** | 4456 | 98.7 % | | **C10:1** | 4456 | 99.5 % | **C18:2** | 4456 | 99.7 % | | **C10:2** | 4456 | 99.6 % | **C18OH** | 4456 | 99.3 % | | **C12** | 4456 | 99.3 % | **C18:10H** | 4456 | 98.0 % | | **C12:1** | 4456 | 99.3 % | | | | **Agreement between NeoBase2 and NeoBase analytes with 1% and 10% cutoff** | Analyte | N | Overall Agreement | | --- | --- | --- | | **Cit** | 4456 | 98.2 % | | **C0 (10% cutoff)** | 4456 | 94.1 % | | **C2** | 4456 | 99.2 % | | **C3** | 4456 | 99.0 % | | **C16** | 4456 | 99.3 % | | **C18** | 4456 | 99.3 % | | **C18:1** | 4456 | 99.5 % | 34 {34} # Summary of screening performance for different disorders | Amino acid disorders (AA): N=37 | | | | | | --- | --- | --- | --- | --- | | Disorder | Abbreviation | Number of positive specimens | Detected with NeoBase 2 Study Cutoffs | Detected with NeoBase Study Cutoffs | | Arginemia | ARG | 2 | 2 | 2 | | Argininosuccinic acidemia | ASA | 3 | 3 | 3 | | Benign hyperphenylalaninemia | H-PHE | 4 | 4 | 4 | | Citrullinemia type I | CIT I | 7 | 7 | 7 | | Classic phenylketonuria | PKU | 4 | 4 | 4 | | Disorders of biopterin defect in cofactor biosynthesis | BIOPT-BS | 1 | 1 | 1 | | Homocystinuria | HCY | 1 | 1 | 1 | | Hypermethioninemia | MET | 2 | 2 | 2 | | Maple syrup urine disease | MSUD | 6 | 6 | 6 | | Ornithine transcarbamylase deficiency | OTCD | 3 | 3 | 3 | | Tyrosinemia, type I | TYR I | 2 | 2 | 2 | | Tyrosinemia, type II | TYR II | 1 | 1 | 1 | | Tyrosinemia, type III | TYR III | 1 | 1 | 1 | | Fatty acid oxidation disorders (FAO): N=26 | | | | | | Disorder | Abbreviation | Number of positive specimens | Detected with NeoBase2 Study Cutoffs | Detected with NeoBase Study Cutoffs | | Carnitine palmitoyltransferase | CPT II | 1 | 1 | 1 | | Carnitine uptake defect | CUD | 4 | 4 | 4 | | Long-chain L-3-hydroxy acyl-CoA dehydrogenase deficiency | LCHAD | 4 | 4 | 4 | | Medium-chain acyl-CoA dehydrogenase deficiency | MCAD | 5 | 5 | 5 | | Short-chain acyl-CoA dehydrogenase deficiency | SCAD | 4 | 4 | 4 | | Trifunctional protein deficiency | TFP | 2 | 2 | 2 | | Very long-chain acyl-CoA dehydrogenase deficiency | VLCAD | 6 | 6 | 6 | 35 {35} | Organic acid conditions (OA): N=28 | | | | | | --- | --- | --- | --- | --- | | Disorder | Abbreviation | Number of positive specimens | Detected with NeoBase2 Study Cutoffs | Detected with NeoBase Study Cutoffs | | 2-Methylbutyrylglycinuria | 2MBG | 2 | 2 | 2 | | 3-Methylcrotonyl-CoA carboxylase deficiency | 3MCC | 4 | 4 | 4 | | 3-Methylglutaconic academia type I | 3MGA | 1 | 1 | 1 | | Glutaric acidemia type I | GA I | 6 | 6 | 6 | | Holocarboxylase synthetase deficiency | MCD | 1 | 1 | 1 | | Isobutyrylglycinuria | IBG | 1 | 1 | 1 | | Isovaleric acidemia | IVA | 4 | 4 | 4 | | Methylmalonic acidemia (methylmalonyl-CoA mutase) | MUT | 3 | 3 | 3 | | Methylmalonic acidemia (cbl disorders) | cblA, cblB | 1 | 1 | 1 | | Propionic acidemia | PROP | 5 | 5 | 5 | | Other disorders: N=8 | | | | | | Disorder | Abbreviation | Number of positive specimens | Detected with NeoBase2 Study Cutoffs | Detected with NeoBase Study Cutoffs | | Adenosine Deaminase Severe Combined Immunodeficiency | ADA-SCID | 4 | 4 | NA^{†} | | X-linked Adrenoleukodystrophy | X-ALD | 4 | 4 | NA^{†} | † - not tested in Neobase instrument 2x2 tables using 99.5th percentile cutoff from site 1 | Ala | NEOBASE | | | | | --- | --- | --- | --- | --- | | | | Positive | Negative | Total | | NEOBASE2 | Positive | 9 | 0 | 9 | | | Negative | 8 | 1762 | 1770 | | | Total | 17 | 1762 | 1779 | 36 {36} | Arg | NEOBASE | | | | | --- | --- | --- | --- | --- | | | | Positive | Negative | Total | | NEOBASE2 | Positive | 9 | 0 | 9 | | | Negative | 4 | 1766 | 1770 | | | Total | 13 | 1766 | 1779 | | C0 | NEOBASE | | | | | --- | --- | --- | --- | --- | | | | Positive | Negative | Total | | NEOBASE2 | Positive | 6 | 3 | 9 | | | Negative | 2 | 1768 | 1770 | | | Total | 8 | 1771 | 1779 | | C10 | NEOBASE | | | | | --- | --- | --- | --- | --- | | | | Positive | Negative | Total | | NEOBASE2 | Positive | 6 | 0 | 6 | | | Negative | 5 | 1768 | 1773 | | | Total | 11 | 1768 | 1779 | | C10:1 | NEOBASE | | | | | --- | --- | --- | --- | --- | | | | Positive | Negative | Total | | NEOBASE2 | Positive | 8 | 3 | 11 | | | Negative | 1 | 1767 | 1768 | | | Total | 9 | 1770 | 1779 | | C10:2 | NEOBASE | | | | | --- | --- | --- | --- | --- | | | | Positive | Negative | Total | | NEOBASE2 | Positive | 1 | 4 | 5 | | | Negative | 0 | 1774 | 1774 | | | Total | 1 | 1778 | 1779 | | C12 | NEOBASE | | | | | --- | --- | --- | --- | --- | | | | Positive | Negative | Total | | NEOBASE2 | Positive | 9 | 2 | 11 | | | Negative | 3 | 1765 | 1768 | | | Total | 12 | 1767 | 1779 | 37 {37} | C12:1 | NEOBASE | | | | | --- | --- | --- | --- | --- | | | | Positive | Negative | Total | | NEOBASE2 | Positive | 9 | 1 | 10 | | | Negative | 2 | 1767 | 1769 | | | Total | 11 | 1768 | 1779 | | C14 | NEOBASE | | | | | --- | --- | --- | --- | --- | | | | Positive | Negative | Total | | NEOBASE2 | Positive | 10 | 1 | 11 | | | Negative | 2 | 1766 | 1768 | | | Total | 12 | 1767 | 1779 | | C14:1 | NEOBASE | | | | | --- | --- | --- | --- | --- | | | | Positive | Negative | Total | | NEOBASE2 | Positive | 8 | 3 | 11 | | | Negative | 2 | 1766 | 1768 | | | Total | 10 | 1769 | 1779 | | C14:2 | NEOBASE | | | | | --- | --- | --- | --- | --- | | | | Positive | Negative | Total | | NEOBASE2 | Positive | 8 | 1 | 9 | | | Negative | 4 | 1766 | 1770 | | | Total | 12 | 1767 | 1779 | | C14OH | NEOBASE | | | | | --- | --- | --- | --- | --- | | | | Positive | Negative | Total | | NEOBASE2 | Positive | 8 | 0 | 8 | | | Negative | 6 | 1765 | 1771 | | | Total | 14 | 1765 | 1779 | | C16 | NEOBASE | | | | | --- | --- | --- | --- | --- | | | | Positive | Negative | Total | | NEOBASE2 | Positive | 2 | 0 | 2 | | | Negative | 1 | 1776 | 1777 | | | Total | 3 | 1776 | 1779 | 38 {38} | C16:1 | NEOBASE | | | | | --- | --- | --- | --- | --- | | | | Positive | Negative | Total | | NEOBASE2 | Positive | 6 | 1 | 7 | | | Negative | 2 | 1770 | 1772 | | | Total | 8 | 1771 | 1779 | | C16:1OH | NEOBASE | | | | | --- | --- | --- | --- | --- | | | | Positive | Negative | Total | | NEOBASE2 | Positive | 8 | 2 | 10 | | | Negative | 2 | 1767 | 1769 | | | Total | 10 | 1769 | 1779 | | C16OH | NEOBASE | | | | | --- | --- | --- | --- | --- | | | | Positive | Negative | Total | | NEOBASE2 | Positive | 6 | 0 | 6 | | | Negative | 3 | 1770 | 1773 | | | Total | 9 | 1770 | 1779 | | C18 | NEOBASE | | | | | --- | --- | --- | --- | --- | | | | Positive | Negative | Total | | NEOBASE2 | Positive | 3 | 3 | 6 | | | Negative | 1 | 1772 | 1773 | | | Total | 4 | 1775 | 1779 | | C18:1 | NEOBASE | | | | | --- | --- | --- | --- | --- | | | | Positive | Negative | Total | | NEOBASE2 | Positive | 2 | 3 | 5 | | | Negative | 7 | 1767 | 1774 | | | Total | 9 | 1770 | 1779 | | C18:1OH | NEOBASE | | | | | --- | --- | --- | --- | --- | | | | Positive | Negative | Total | | NEOBASE2 | Positive | 7 | 1 | 8 | | | Negative | 2 | 1769 | 1771 | | | Total | 9 | 1770 | 1779 | 39 {39} | C18:2 | NEOBASE | | | | | --- | --- | --- | --- | --- | | | | Positive | Negative | Total | | NEOBASE2 | Positive | 14 | 0 | 14 | | | Negative | 4 | 1761 | 1765 | | | Total | 18 | 1761 | 1779 | | C18OH | NEOBASE | | | | | --- | --- | --- | --- | --- | | | | Positive | Negative | Total | | NEOBASE2 | Positive | 6 | 4 | 10 | | | Negative | 0 | 1769 | 1769 | | | Total | 6 | 1773 | 1779 | | C3 | NEOBASE | | | | | --- | --- | --- | --- | --- | | | | Positive | Negative | Total | | NEOBASE2 | Positive | 6 | 0 | 6 | | | Negative | 2 | 1771 | 1773 | | | Total | 8 | 1771 | 1779 | | C3DC\C4OH | NEOBASE | | | | | --- | --- | --- | --- | --- | | | | Positive | Negative | Total | | NEOBASE2 | Positive | 9 | 11 | 20 | | | Negative | 4 | 1755 | 1759 | | | Total | 13 | 1766 | 1779 | | C4 | NEOBASE | | | | | --- | --- | --- | --- | --- | | | | Positive | Negative | Total | | NEOBASE2 | Positive | 10 | 2 | 12 | | | Negative | 0 | 1767 | 1767 | | | Total | 10 | 1769 | 1779 | | C4DC\C5OH | NEOBASE | | | | | --- | --- | --- | --- | --- | | | | Positive | Negative | Total | | NEOBASE2 | Positive | 7 | 0 | 7 | | | Negative | 0 | 1772 | 1772 | | | Total | 7 | 1772 | 1779 | 40 {40} | C5 | NEOBASE | | | | | --- | --- | --- | --- | --- | | | | Positive | Negative | Total | | NEOBASE2 | Positive | 10 | 3 | 13 | | | Negative | 1 | 1765 | 1766 | | | Total | 11 | 1768 | 1779 | | C5:1 | NEOBASE | | | | | --- | --- | --- | --- | --- | | | | Positive | Negative | Total | | NEOBASE2 | Positive | 3 | 3 | 6 | | | Negative | 3 | 1770 | 1773 | | | Total | 6 | 1773 | 1779 | | C5DC\C6OH | NEOBASE | | | | | --- | --- | --- | --- | --- | | | | Positive | Negative | Total | | NEOBASE2 | Positive | 9 | 9 | 18 | | | Negative | 3 | 1758 | 1761 | | | Total | 12 | 1767 | 1779 | | C6 | NEOBASE | | | | | --- | --- | --- | --- | --- | | | | Positive | Negative | Total | | NEOBASE2 | Positive | 9 | 0 | 9 | | | Negative | 1 | 1769 | 1770 | | | Total | 10 | 1769 | 1779 | | C6DC | NEOBASE | | | | | --- | --- | --- | --- | --- | | | | Pos…