NeoBase 2 Non-derivatized MSMS Kit

K193103 · Perkinelmer, Inc. · NQL · Feb 7, 2020 · Clinical Chemistry

Device Facts

• Record ID: K193103
• Device Name: NeoBase 2 Non-derivatized MSMS Kit
• Applicant: Perkinelmer, Inc.
• Product Code: NQL · Clinical Chemistry
• Decision Date: Feb 7, 2020
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 862.1055
• Device Class: Class 2
• Attributes: Pediatric

Intended 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.

Device Story

The NeoBase 2 Non-derivatized MSMS kit is an in vitro diagnostic screening assay for newborns. It uses dried blood spot (DBS) samples collected on filter paper. The assay extracts analytes (amino acids, carnitines, nucleosides, lysophospholipids) using an extraction working solution containing internal standards. Succinylacetone (SA) is derivatized simultaneously with hydrazine to form a stable pyrazole-like product. The extract is analyzed via tandem mass spectrometry (MSMS) in Multiple Reaction Monitoring (MRM) mode using the PerkinElmer QSight 210 MD Screening System. The system measures analyte-specific precursor-product ion transitions. The ratio of analyte signal to internal standard signal is proportional to concentration. Results are used by clinicians as an aid to other medically established procedures for metabolic disorder screening. The device is not for confirmatory or prenatal testing. It requires prescription use and is operated by laboratory personnel.

Clinical Evidence

Clinical study compared QSight 210MD and TQD platforms using 2,530 routine newborn screening specimens. Performance evaluated via percentile cut-offs (1st, 10th, 99th, 99.5th). Study included confirmed positive specimens for amino acid disorders (n=19), fatty acid oxidation disorders (n=12), organic acid conditions (n=16), and other conditions (n=5). Results demonstrate equivalent screening performance between platforms.

Technological Characteristics

The system uses tandem mass spectrometry (MSMS) with Multiple Reaction Monitoring (MRM). It utilizes the PerkinElmer QSight 210 MD Screening System, including the QSight 210 MD Mass Spectrometer, QSight HC Autosampler MD, and QSight Binary Pump MD. Reagents include extraction solutions and isotope-labeled internal standards. The assay is designed for 960 tests per kit using Whatman no. 903 filter paper. Software includes Simplicity 3Q MD and PerkinElmer MSMS Workstation.

Indications for Use

Indicated for newborn screening of metabolic disorders using dried heel prick blood specimens. Evaluates concentrations of amino acids, succinylacetone, free carnitine, acylcarnitines, nucleosides, and lysophospholipids to aid in identifying metabolic conditions.

Regulatory Classification

Identification

A newborn screening test system for amino acids, free carnitine, and acylcarnitines using tandem mass spectrometry is a device that consists of stable isotope internal standards, control materials, extraction solutions, flow solvents, instrumentation, software packages, and other reagents and materials. The device is intended for the measurement and evaluation of amino acids, free carnitine, and acylcarnitine concentrations from newborn whole blood filter paper samples. The quantitative analysis of amino acids, free carnitine, and acylcarnitines and their relationship with each other provides analyte concentration profiles that may aid in screening newborns for one or more inborn errors of amino acid, free carnitine, and acyl-carnitine metabolism.

Special Controls

*Classification.* Class II (special controls). The special control is FDA's guidance document entitled “Class II Special Controls Guidance Document: Newborn Screening Test Systems for Amino Acids, Free Carnitine, and Acylcarnitines Using Tandem Mass Spectrometry.” See § 862.1(d) for the availability of this guidance document.

Predicate Devices

• NeoBase 2 Non-derivatized MSMS kit (K173568)

Submission Summary (Full Text)

{0}------------------------------------------------

Image /page/0/Picture/0 description: The image shows the logo of the U.S. Food & Drug Administration (FDA). On the left is the Department of Health & Human Services logo. To the right of that is the FDA logo, which is a blue square with the letters "FDA" in white. To the right of the blue square is the text "U.S. FOOD & DRUG ADMINISTRATION" in blue.

February 7, 2020

PerkinElmer Inc. Eva Nalian Sr. Manager Regulatory Affairs 940 Winter Street Waltham, MA 02451

Re: K193103

Trade/Device Name: NeoBase 2 Non-derivatized MSMS Kit Regulation Number: 21 CFR 862.1055 Regulation Name: Newborn Screening Test System for Amino Acids, Free Carnitine, and Acylcarnitines Using Tandem Mass Spectrometry Regulatory Class: Class II Product Code: NOL Dated: November 7, 2019 Received: November 8, 2019

Dear Eva Nalian:

We have reviewed vour Section 510(k) premarket notification of intent to market the device referenced above and have determined the device is substantially equivalent (for the indications for use stated in the enclosure) to legally marketed predicate devices marketed in interstate commerce prior to May 28, 1976, the enactment date of the Medical Device Amendments, or to devices that have been reclassified in accordance with the provisions of the Federal Food, Drug, and Cosmetic Act (Act) that do not require approval of a premarket approval application (PMA). You may, therefore, market the device, subject to the general controls provisions of the Act. Although this letter refers to your product as a device, please be aware that some cleared products may instead be combination products. The 510(k) Premarket Notification Database located at https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfpmn/pmn.cfm identifies combination product submissions. The general controls provisions of the Act include requirements for annual registration, listing of devices, good manufacturing practice, labeling, and prohibitions against misbranding and adulteration. Please note: CDRH does not evaluate information related to contract liability warranties. We remind you, however, that device labeling must be truthful and not misleading.

If your device is classified (see above) into either class II (Special Controls) or class III (PMA), it may be subject to additional controls. Existing major regulations affecting your device can be found in the Code of Federal Regulations, Title 21, Parts 800 to 898. In addition, FDA may publish further announcements concerning your device in the Federal Register.

Please be advised that FDA's issuance of a substantial equivalence determination does not mean that FDA has made a determination that your device complies with other requirements of the Act or any Federal

{1}------------------------------------------------

statutes and regulations administered by other Federal agencies. You must comply with all the Act's requirements, including, but not limited to: registration and listing (21 CFR Part 807); labeling (21 CFR Part 801 and Part 809); medical device reporting of medical device-related adverse events) (21 CFR 803) for devices or postmarketing safety reporting (21 CFR 4, Subpart B) for combination products (see https://www.fda.gov/combination-products/guidance-regulatory-information/postmarketing-safety-reportingcombination-products); good manufacturing practice requirements as set forth in the quality systems (QS) regulation (21 CFR Part 820) for devices or current good manufacturing practices (21 CFR 4, Subpart A) for combination products; and, if applicable, the electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR 1000-1050.

Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21 CFR Part 807.97). For questions regarding the reporting of adverse events under the MDR regulation (21 CFR Part 803), please go to https://www.fda.gov/medical-device-safety/medical-device-reportingmdr-how-report-medical-device-problems.

For comprehensive regulatory information about medical devices and radiation-emitting products, including information about labeling regulations, please see Device Advice (https://www.fda.gov/medicaldevices/device-advice-comprehensive-regulatory-assistance) and CDRH Learn (https://www.fda.gov/training-and-continuing-education/cdrh-learn). Additionally, you may contact the Division of Industry and Consumer Education (DICE) to ask a question about a specific regulatory topic. See the DICE website (https://www.fda.gov/medical-device-advice-comprehensive-regulatoryassistance/contact-us-division-industry-and-consumer-education-dice) for more information or contact DICE by email ([e-mail]) or phone [phone] or 301-796-7100).

Sincerely.

Kellie B. Kelm, Ph.D. Acting Director Division Director of Chemistry and Toxicology Devices OHT7: Office of In Vitro Diagnostics and Radiological Health Office of Product Evaluation and Ouality Center for Devices and Radiological Health

Enclosure

{2}------------------------------------------------

# Indications for Use

510(k) Number (if known)

K193103

Device Name NeoBase™ 2 Non-derivatized MSMS kit

#### Indications for Use (Describe)

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 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 NeoBase 2 Non-derivatized MSMS kit.

| ANALYTE NAME | ABBREVIATION |
|-------------------------------------------------|----------------|
| Amino acids | |
| Alanine | Ala |
| Arginine | Arg |
| Argininosuccinic acid | Asa |
| Citrulline | Cit |
| Glutamine\Lysine 1 | Gln\Lys |
| Glutamic acid | Glu |
| Glycine | Gly |
| Leucine\Isoleucine\Hydroxyproline 1 | Leu\lle\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 1 | C3DC\C4OH |
| Butyrylcarnitine | C4 |
| Methylmalonyl\3-Hydroxy-isovalerylcarnitine 1 | C4DC\C5OH |
| Isovalerylcarnitine | C5 |
| Tiglylcarnitine | C5:1 |
| Glutarylcarnitine\3-Hydroxy-hexanoylcarnitine 1 | C5DC\C6OH |
| Hexanoylcarnitine | C6 |
| Adipylcarnitine | C6DC |

1 Analytes in these rows are either isomers or isobars and cannot be distinguished in the tandem mass spectrometry experiment.

{3}------------------------------------------------

Table 1 (continued): Analytes measured by the NeoBase 2 Non-derivatized MSMS kit.

| 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\<br>Heptadecanoylcarnitine 1 | 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 |

1 Analytes in these rows are either isomers or isobars and cannot be distinguished in the tandem mass spectrometry experiment.

{4}------------------------------------------------

Type of Use (Select one or both, as applicable)

> Prescription Use (Part 21 CFR 801 Subpart D)

__ Over-The-Counter Use (21 CFR 801 Subpart C)

#### CONTINUE ON A SEPARATE PAGE IF NEEDED.

This section applies only to requirements of the Paperwork Reduction Act of 1995.

#### *DO NOT SEND YOUR COMPLETED FORM TO THE PRA STAFF EMAIL ADDRESS BELOW.*

The burden time for this collection of information is estimated to average 79 hours per response, including the time to review instructions, search existing data sources, gather and maintain the data needed and complete and review the collection of information. Send comments regarding this burden estimate or any other aspect of this information collection, including suggestions for reducing this burden, to:

> Department of Health and Human Services Food and Drug Administration Office of Chief Information Officer Paperwork Reduction Act (PRA) Staff [e-mail]

"An agency may not conduct or sponsor, and a person is not required to respond to, a collection of information unless it displays a currently valid OMB number."

FORM FDA 3881 (7/17)

Page 1 of 1

PSC Publishing Services [phone] EF

{5}------------------------------------------------

Image /page/5/Picture/1 description: The image shows the logo for PerkinElmer. The logo features the company name in a bold, sans-serif font, with the "Perkin" part in black and the "Elmer" part in blue. Above the name is a stylized blue graphic element that resembles an abstract shape. Below the name is the company's slogan, "For the Better," also in blue.

# 510(k) Summary

This summary of safety and effectiveness information is supplied in accordance with the requirements of SMDA 1990 and 21 CFR 807.92.

The assigned number is _K193103

| Submitted by: | PerkinElmer, Inc.<br>940 Winter Street<br>Waltham MA 02451 |
|-------------------|---------------------------------------------------------------------------------------------------------------------|
| Contact Person: | Eva Nalian<br>Tel: [phone] |
| Trade Name: | NeoBase 2 Non-derivatized MSMS kit |
| Common Name: | Newborn screening test system for amino acids, free<br>carnitine, and acylcarnitines using tandem mass spectrometry |
| Regulation: | 21 CFR 862.1055 |
| Classification: | II |
| Panel: | 75 Chemistry |
| Product Code: | NQL |
| Predicate device: | NeoBase 2 Non-derivatized MSMS kit (K173568) |

{6}------------------------------------------------

Image /page/6/Picture/1 description: The image shows the logo for PerkinElmer. The logo consists of a blue stylized "P" shape with a triangle cut out of the top right corner. Below the symbol, the name "PerkinElmer" is written in a combination of black and gray text. Underneath the name, the phrase "For the Better" is written in a smaller font size.

## 1. 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 - 1 vial
- 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.

Instruments used:

- . QSight® 210 MD Screening System is comprised of:
- QSight® 210 MD Mass Spectrometer
- QSight® HC Autosampler MD
- QSight® Binary Pump MD ●
- Simplicity™ 3Q MD Software
- PerkinElmer MSMS Workstation software ●

{7}------------------------------------------------

Image /page/7/Picture/1 description: The image shows the logo for PerkinElmer. The logo features a stylized blue "P" shape with a pointed end, positioned above the company name. The text "PerkinElmer" is written in a bold, sans-serif font, with the "E" in "Elmer" slightly larger and in a lighter shade of blue. Below the company name, the tagline "For the Better" is written in a smaller, italicized font, also in blue.

### 2. Intended Use:

The NeoBase 2 Non-derivatized MSMS kit is intended for the measurement and evaluation of amino acid, succinylacetone, free carnitine, 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 NeoBase 2 Non-derivatized MSMS kit.

| ANALYTE NAME | ABBREVIATION |
|-------------------------------------------------|----------------|
| Amino acids | |
| Alanine | Ala |
| Arginine | Arg |
| Argininosuccinic acid | Asa |
| Citrulline | Cit |
| Glutamine\Lysine 1 | Gln\Lys |
| Glutamic acid | Glu |
| Glycine | Gly |
| Leucine\Isoleucine\Hydroxyproline 1 | Leu\lle\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 1 | C3DC\C4OH |
| Butyrylcarnitine | C4 |
| Methylmalonyl\3-Hydroxy-isovalerylcarnitine 1 | C4DC\C5OH |
| Isovalerylcarnitine | C5 |
| Tiglylcarnitine | C5:1 |
| Glutarylcarnitine\3-Hydroxy-hexanoylcarnitine 1 | C5DC\C6OH |
| Hexanoylcarnitine | C6 |
| Adipylcarnitine | C6DC |

1 Analytes in these rows are either isomers or isobars and cannot be distinguished in the tandem mass spectrometry experiment.

{8}------------------------------------------------

Image /page/8/Picture/1 description: The image shows the logo for PerkinElmer. The logo features a blue abstract symbol above the company name. The symbol consists of a vertical bar and a right-pointing triangle. Below the company name is the tagline "For the Better" in a smaller, italicized font.

Table 1 (continued): Analytes measured by the NeoBase 2 Non-derivatized MSMS kit.

| 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\ | C16:1OH\C17 |
| Heptadecanoylcarnitine 1 | 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 |

1 Analytes in these rows are either isomers or isobars and cannot be distinguished in the tandem mass spectrometry experiment.

## 3. Substantial Equivalency:

{9}------------------------------------------------

Image /page/9/Picture/1 description: The image shows the logo for PerkinElmer. The logo consists of a blue abstract shape resembling a stylized "P" and a right-pointing arrow. Below the shape, the text "PerkinElmer" is written in a bold, sans-serif font. Underneath "PerkinElmer", the tagline "For the Better" is written in a smaller, italicized, blue font.

| Characteristics | Proposed Device | Predicate (K173568) |
|--------------------------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Intended Use/<br>Indications for Use | The NeoBase™ 2 Non-derivatized MSMS kit is<br>intended for the measurement and evaluation of<br>amino acid, succinylacetone, free carnitine,<br>acylcarnitine, nucleoside and lysophospholipid<br>concentrations with a tandem mass spectrometer<br>from newborn heel prick blood specimens dried on<br>filter paper. Quantitative analysis of these analytes<br>and their relationship with each other is intended to<br>provide analyte concentration profiles that may aid<br>in screening newborns for metabolic disorders. | Same |
| Test Principle | Analytes in sample are measured by tandem mass<br>spectrometry through analyte-specific mass<br>transitions appropriate for each type of analyte. The<br>extracted analytes are measured for set time<br>periods and compared to the signal intensities<br>produced by the corresponding isotope- labeled<br>internal standards. The concentrations are<br>determined by comparing the signal intensities of<br>the known standards to the measured analytes. | Same |
| Disorders Screened | Amino-, organic-, and fatty acid metabolic disorders | Same |
| Analytes Measured | Amino acids, free carnitine, acylcarnitines,<br>succinylacetone, nucleosides, and lysophospholipids | Same |
| Instrument / Software<br>Platform | PerkinElmer QSight 210MD Screening System: QSight® Waters TQD instrument with MassLynx v4.1<br>HC Autosampler MD,QSight® Binary Pump MD,<br>Simplicity™ 3Q MD Software, PerkinElmer MSMS<br>Workstation Software | firmware, with Waters 1525 sample pump,<br>with Waters 2777c autosampler, with Waters<br>NeoLynx v4.1 software and with the<br>PerkinElmer MSMS Workstation Software |
| Sample Type | Punch from dried blood spot specimen | Same |
| Calibrators | Internal calibration using several isotopically labeled<br>standards, included as dried material in vials.<br>Internal standards must be reconstituted with<br>extraction solution prior to their use. | Same |
| Throughput | Ninety-six tests per microtiter<br>plate. Multiple plates can be analyzed | Same |

{10}------------------------------------------------

Image /page/10/Picture/1 description: The image shows the logo for PerkinElmer. The logo consists of the company name in a bold, sans-serif font, with the words "For the Better" in a smaller, italicized font underneath. Above the company name is a blue graphic element that resembles a stylized "P" or an arrow pointing to the right. The overall design is clean and modern, conveying a sense of innovation and progress.

The proposed device and predicate device utilize similar design shown to produce equivalent screening performance in a clinical setting. Both devices are intended for the measurement and evaluation of multiple metabolite concentrations from newborn heel prick blood samples 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.

## 4. Summary of the Studies:

#### Precision:

The precision was determined in accordance with CLSI document EP05-A3. The repeatability and withinlaboratory variation for NeoBase 2 Non-derivatized MSMS kit is based on 80 determinations: 40 plates measured over 20 working days, each plate having 2 replicates per sample. One QSight was used. Betweenlot 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 is based on altogether 50 determinations: 10 plates were measured with two QSights over 5 working days, each plate having 5 replicates per sample. The results are presented in the table below.

Repeatability, within-laboratory, between-lot, between-instrument and total variation determined for the NeoBase 2 Non-derivatized MSMS kit using QSight:

| Sample | Total<br>mean<br>μmol/L | Repeatability | | Within-Lab | | Between-lot | | Between-<br>instrument | | Total<br>Variation | |
|--------|-------------------------|---------------|-----|------------|-----|-------------|-----|------------------------|------|--------------------|-----|
| | | SD | CV% | SD | CV% | SD | CV% | SD | CV% | SD | CV% |
| 1 | 161 | 11 | 6.5 | 17 | 9.8 | 4.1 | 2.8 | 19 | 12 | 26 | 16 |
| 2 | 361 | 21 | 5.5 | 27 | 7.1 | 6.2 | 1.8 | 17 | 4.6 | 32 | 9.0 |
| 3 | 414 | 24 | 5.4 | 30 | 6.8 | 4.2 | 1.0 | 3.4 | 0.84 | 30 | 7.3 |
| 4 | 518 | 28 | 5.3 | 34 | 6.3 | 15 | 3.1 | 0.04 | 0.01 | 37 | 7.2 |

#### Ala

Arg

| Sample | Total<br>mean<br>μmol/L | | Repeatability | | Within-Lab | | Between-lot | | Between-<br>instrument | | Total<br>Variation | |
|--------|-------------------------|------|---------------|------|------------|------|-------------|-------|------------------------|------|--------------------|--|
| | | SD | CV% | SD | CV% | SD | CV% | SD | CV% | SD | CV% | |
| 1 | 7.5 | 0.45 | 6.0 | 0.62 | 8.3 | 0.47 | 6.8 | <0.01 | <0.01 | 0.78 | 10 | |
| 2 | 23 | 1.3 | 5.7 | 1.7 | 7.3 | 0.11 | 0.49 | <0.01 | <0.01 | 1.7 | 7.6 | |
| 3 | 69 | 3.4 | 4.9 | 3.7 | 5.4 | 1.7 | 2.6 | <0.01 | <0.01 | 4.1 | 5.9 | |
| 4 | 157 | 5.2 | 3.3 | 8.0 | 5.0 | 5.2 | 3.5 | 1.1 | 0.67 | 9.6 | 6.1 | |

{11}------------------------------------------------

Image /page/11/Picture/1 description: The image shows the logo for PerkinElmer. The logo features a blue stylized "P" with a triangle cut out of the top right corner, positioned above the company name "PerkinElmer" in a bold, sans-serif font. Below the company name is the tagline "For the Better" in a smaller, italicized font, also in blue.

| Asa1 | | | | | | | | | | | | | |
|--------|-------------------------|---------------|-----|------------|-----|-------------|------|------------------------|-----|--------------------|-----|--|--|
| Sample | Total<br>mean<br>μmol/L | Repeatability | | Within-Lab | | Between-lot | | Between-<br>instrument | | Total<br>Variation | | | |
| | | SD | CV% | SD | CV% | SD | CV% | SD | CV% | SD | CV% | | |
| 1 | 0.31 | 0.06 | 29 | 0.09 | 43 | <0.01 | 0.74 | 0.03 | 14 | 0.10 | 32 | | |
| 2 | 2.2 | 0.14 | 6.7 | 0.24 | 11 | 0.07 | 3.1 | 0.10 | 4.3 | 0.27 | 12 | | |
| 3 | 8.1 | 0.39 | 5.0 | 0.76 | 9.9 | 0.44 | 5.3 | 0.66 | 7.8 | 1.1 | 14 | | |
| 4 | 21 | 0.74 | 3.6 | 1.7 | 8.1 | 0.95 | 4.5 | 1.7 | 7.8 | 2.6 | 12 | | |
| 5 | 57 | 2.3 | 3.8 | 5.6 | 9.2 | 2.1 | 4.6 | 5.7 | 8.7 | 8.2 | 14 | | |

¹ Asa is measured as a total concentration of Asa and its anhydrides.

## Cit

| Sample | Total<br>mean<br>μmol/L | Repeatability | | Within-Lab | | Between-lot | | Between-<br>instrument | | Total<br>Variation | |
|--------|-------------------------|---------------|-----|------------|-----|-------------|-----|------------------------|-------|--------------------|-----|
| | | SD | CV% | SD | CV% | SD | CV% | SD | CV% | SD | CV% |
| 1 | 10 | 1.1 | 12 | 1.2 | 12 | 0.33 | 3.0 | <0.01 | 0.01 | 1.2 | 12 |
| 2 | 68 | 3.3 | 5.0 | 4.3 | 6.5 | 2.1 | 3.1 | 1.2 | 1.7 | 5.0 | 7.3 |
| 3 | 202 | 10 | 5.1 | 11 | 5.3 | 5.4 | 2.7 | <0.01 | <0.01 | 12 | 5.9 |
| 4 | 470 | 27 | 5.8 | 29 | 6.3 | 11 | 2.3 | 10 | 2.1 | 33 | 6.9 |
| 5 | 957 | 50 | 5.1 | 55 | 5.7 | 27 | 3.0 | 16 | 1.6 | 64 | 6.7 |

## Gln\Lys

| Sample | Total<br>mean<br>μmol/L | Repeatability | | Within-Lab | | Between-lot | | Between-<br>instrument | | Total<br>Variation | |
|--------|-------------------------|---------------|-----|------------|-----|-------------|------|------------------------|-------|--------------------|-----|
| | | SD | CV% | SD | CV% | SD | CV% | SD | CV% | SD | CV% |
| 1 | 43 | 2.8 | 5.9 | 3.7 | 7.8 | 1.0 | 2.5 | 2.9 | 7.2 | 4.8 | 11 |
| 2 | 487 | 24 | 4.6 | 30 | 5.7 | 15 | 3.3 | 0.02 | <0.01 | 34 | 6.9 |
| 3 | 675 | 35 | 4.9 | 43 | 6.0 | 3.3 | 0.52 | <0.01 | <0.01 | 43 | 6.4 |
| 4 | 1064 | 52 | 4.6 | 65 | 5.7 | 37 | 3.7 | 13 | 1.2 | 75 | 7.1 |
| 5 | 2274 | 94 | 3.9 | 136 | 5.7 | 20 | 0.90 | 0.13 | 0.01 | 138 | 6.1 |

Gly

| Sample | Total<br>mean<br>μmol/L | Repeatability | | Within-Lab | | Between-lot | | Between-<br>instrument | | Total<br>Variation | |
|--------|-------------------------|---------------|-----|------------|-----|-------------|-----|------------------------|-------|--------------------|-----|
| | | SD | CV% | SD | CV% | SD | CV% | SD | CV% | SD | CV% |
| 1 | 247 | 16 | 6.6 | 24 | 9.8 | 7.9 | 3.0 | 3.9 | 1.6 | 25 | 10 |
| 2 | 324 | 19 | 6.3 | 23 | 7.6 | 6.6 | 1.9 | 4.7 | 1.5 | 25 | 7.6 |
| 3 | 524 | 32 | 6.3 | 41 | 8.1 | 6.1 | 1.1 | 0.01 | <0.01 | 42 | 8.0 |
| 4 | 930 | 52 | 5.7 | 82 | 8.9 | 26 | 2.8 | 19 | 2.1 | 88 | 9.5 |

### Leu\lle\Pro-OH

| Sample | Total<br>mean<br>μmol/L | Repeatability | | Within-Lab | Between-lot | | Between-<br>instrument | | Total<br>Variation | | |
|--------|-------------------------|---------------|-----|------------|-------------|------|------------------------|-------|--------------------|-----|-----|
| | | SD | CV% | SD | CV% | SD | CV% | SD | CV% | SD | CV% |
| 1 | 58 | 2.5 | 4.3 | 3.2 | 5.5 | 0.54 | 0.87 | <0.01 | <0.01 | 3.2 | 5.6 |

{12}------------------------------------------------

Image /page/12/Picture/0 description: The image shows the logo for PerkinElmer. The logo consists of a blue stylized "P" above the name "PerkinElmer" in black. Below the name is the tagline "For the Better" in a smaller, italicized font.

| 2 | 202 | 9.0 | 4.5 | 11 | 5.7 | 4.7 | 2.3 | <0.01 | <0.01 | 12 | 6.2 |
|---|------|-----|-----|----|-----|-----|------|-------|-------|----|-----|
| 3 | 350 | 16 | 4.4 | 16 | 4.4 | 2.7 | 0.80 | <0.01 | <0.01 | 16 | 4.6 |
| 4 | 656 | 31 | 4.7 | 33 | 4.9 | 17 | 2.6 | 0.03 | 0.01 | 37 | 5.6 |
| 5 | 1121 | 45 | 3.9 | 54 | 4.7 | 27 | 2.5 | <0.01 | <0.01 | 60 | 5.4 |

Met

| Sample | Total<br>mean<br>μmol/L | Repeatability | | Within-Lab | | Between-lot | | Between-<br>instrument | | Total<br>Variation | |
|--------|-------------------------|---------------|-----|------------|-----|-------------|-----|------------------------|-------|--------------------|-----|
| | | SD | CV% | SD | CV% | SD | CV% | SD | CV% | SD | CV% |
| 1 | 2.2 | 0.28 | 18 | 0.28 | 18 | 0.26 | 8.3 | 0.21 | 11 | 0.43 | 20 |
| 2 | 51 | 2.6 | 5.1 | 3.1 | 6.2 | 1.9 | 3.7 | 0.58 | 1.1 | 3.7 | 7.3 |
| 3 | 155 | 8.2 | 5.4 | 9.1 | 5.9 | 2.3 | 1.5 | <0.01 | <0.01 | 9.4 | 6.1 |
| 4 | 369 | 19 | 5.0 | 23 | 6.3 | 9.8 | 2.7 | 4.2 | 1.1 | 26 | 6.9 |
| 5 | 696 | 26 | 3.7 | 37 | 5.2 | 14 | 2.0 | 0.01 | <0.01 | 40 | 5.7 |

Orn

| Sample | Total<br>mean<br>μmol/L | Repeatability | | Within-Lab | | Between-lot | | Between-<br>instrument | | Total<br>Variation | |
|--------|-------------------------|---------------|-----|------------|-----|-------------|-----|------------------------|-------|--------------------|-----|
| | | SD | CV% | SD | CV% | SD | CV% | SD | CV% | SD | CV% |
| 1 | 29 | 1.9 | 6.7 | 2.9 | 10 | 0.50 | 1.7 | 0.89 | 2.9 | 3.1 | 11 |
| 2 | 109 | 4.1 | 3.8 | 7.0 | 6.4 | 1.5 | 1.4 | 2.0 | 1.8 | 7.4 | 6.8 |
| 3 | 204 | 10 | 4.9 | 12 | 5.7 | 2.3 | 1.2 | <0.01 | <0.01 | 12 | 5.8 |
| 4 | 382 | 14 | 3.8 | 17 | 4.5 | 9.9 | 2.7 | 11 | 2.7 | 23 | 5.9 |

Phe

| Sample | Total mean<br>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 | 5.1 | 1.2 | 5.8 | 0.43 | 1.7 | <0.01 | <0.01 | 1.3 | 5.8 |
| 2 | 127 | 4.3 | 3.4 | 5.6 | 4.5 | 3.5 | 2.7 | 1.2 | 0.94 | 6.7 | 5.3 |
| 3 | 340 | 15 | 4.5 | 16 | 4.8 | 4.7 | 1.4 | <0.01 | <0.01 | 17 | 5.0 |
| 4 | 778 | 35 | 4.5 | 43 | 5.5 | 29 | 3.8 | 7.3 | 0.91 | 52 | 6.7 |
| 5 | 1436 | 39 | 2.6 | 65 | 4.4 | 17 | 1.2 | 23 | 1.6 | 71 | 4.9 |

Pro

| Sample | Total<br>mean<br>μmol/L | Repeatability | | Within-Lab | | Between-lot | | Between-<br>instrument | | Total<br>Variation | |
|--------|-------------------------|---------------|-----|------------|-----|-------------|------|------------------------|-------|--------------------|-----|
| | | SD | CV% | SD | CV% | SD | CV% | SD | CV% | SD | CV% |
| 1 | 40 | 1.8 | 4.6 | 2.2 | 5.6 | 0.57 | 1.4 | <0.01 | <0.01 | 2.3 | 5.7 |
| 2 | 178 | 7.0 | 3.9 | 8.1 | 4.5 | 4.6 | 2.6 | 2.0 | 1.1 | 9.5 | 5.3 |
| 3 | 316 | 14 | 4.4 | 15 | 4.7 | 2.6 | 0.86 | <0.01 | <0.01 | 16 | 4.9 |
| 4 | 596 | 28 | 4.5 | 30 | 4.8 | 16 | 2.8 | 6.9 | 1.1 | 34 | 5.7 |

{13}------------------------------------------------

Image /page/13/Picture/1 description: The image shows the logo for PerkinElmer. The logo consists of the company name in a bold, sans-serif font, with a blue graphic above it. The graphic is a stylized "P" shape with an arrow pointing to the right. Below the company name is the tagline "For the Better" in a smaller, italicized font.

| Tyr | | | | | | | | | | | |
|--------|---------------|---------------|-----|------------|-----|-------------|-----|------------------------|-------|--------------------|-----|
| Sample | Total<br>mean | Repeatability | | Within-Lab | | Between-lot | | Between-<br>instrument | | Total<br>Variation | |
| | μmol/L | SD | CV% | SD | CV% | SD | CV% | SD | CV% | SD | CV% |
| 1 | 20 | 1.2 | 6.3 | 1.5 | 7.5 | 0.52 | 2.6 | <0.01 | <0.01 | 1.5 | 7.7 |
| 2 | 109 | 4.1 | 3.9 | 4.7 | 4.5 | 3.6 | 3.3 | 1.4 | 1.3 | 6.1 | 5.6 |
| 3 | 264 | 9.2 | 3.6 | 9.2 | 3.6 | 4.1 | 1.6 | <0.01 | <0.01 | 10 | 3.8 |
| 4 | 586 | 21 | 3.6 | 26 | 4.6 | 15 | 2.6 | 2.4 | 0.39 | 30 | 5.1 |

## Val

| Sample | Total<br>mean<br>μmol/L | Repeatability | | Within-Lab | | Between-lot | | Between-<br>instrument | | Total<br>Variation | |
|--------|-------------------------|---------------|-----|------------|-----|-------------|-----|------------------------|-------|--------------------|-----|
| | | SD | CV% | SD | CV% | SD | CV% | SD | CV% | SD | CV% |
| 1 | 55 | 2.4 | 4.5 | 2.8 | 5.2 | 0.81 | 1.3 | <0.01 | <0.01 | 2.9 | 5.2 |
| 2 | 205 | 8.7 | 4.2 | 11 | 5.4 | 5.6 | 2.8 | 3.6 | 1.8 | 13 | 6.4 |
| 3 | 314 | 15 | 4.8 | 15 | 4.8 | 4.2 | 1.4 | <0.01 | <0.01 | 16 | 5.1 |
| 4 | 540 | 27 | 4.9 | 29 | 5.2 | 15 | 3.0 | 10 | 1.9 | 34 | 6.4 |

# C0

| Sample | Total<br>mean<br>μmol/L | Repeatability | | Within-Lab | | Between-lot | | Between-<br>instrument | | Total<br>Variation | |
|--------|-------------------------|---------------|-----|------------|-----|-------------|------|------------------------|------|--------------------|-----|
| | | SD | CV% | SD | CV% | SD | CV% | SD | CV% | SD | CV% |
| 1 | 8.4 | 0.46 | 5.6 | 0.54 | 6.5 | 0.04 | 0.47 | <0.01 | 0.01 | 0.55 | 6.5 |
| 2 | 42 | 1.8 | 4.4 | 2.1 | 5.0 | 1.5 | 3.7 | 1.1 | 2.6 | 2.8 | 6.7 |
| 3 | 89 | 4.9 | 5.4 | 5.4 | 5.9 | 1.1 | 1.2 | 0.01 | 0.02 | 5.5 | 6.1 |
| 4 | 186 | 8.3 | 4.3 | 11 | 5.5 | 6.7 | 3.7 | 3.0 | 1.6 | 13 | 6.9 |

C2

| Sample | Total<br>mean | Repeatability | | Within-Lab | | Between-lot | | Between-<br>instrument | | Total<br>Variation | |
|--------|---------------|---------------|-----|------------|-----|-------------|------|------------------------|-------|--------------------|-----|
| | μmol/L | SD | CV% | SD | CV% | SD | CV% | SD | CV% | SD | CV% |
| 1 | 3.6 | 0.17 | 4.8 | 0.21 | 5.7 | 0.09 | 2.5 | <0.01 | <0.01 | 0.22 | 6.3 |
| 2 | 12 | 0.58 | 4.7 | 0.72 | 5.9 | 0.47 | 3.9 | 0.06 | 0.47 | 0.87 | 7.1 |
| 3 | 18 | 0.87 | 4.7 | 0.91 | 4.9 | 0.09 | 0.49 | <0.01 | <0.01 | 0.91 | 5.0 |
| 4 | 30 | 1.3 | 4.3 | 1.5 | 4.9 | 0.66 | 2.2 | <0.01 | <0.01 | 1.7 | 5.5 |

СЗ

| Sample | Total<br>mean<br>μmol/L | Repeatability | | Within-Lab | | Between-lot | | Between-<br>instrument | | Total<br>Variation | |
|--------|-------------------------|---------------|-----|------------|-----|-------------|-----|------------------------|-------|--------------------|-----|
| | | SD | CV% | SD | CV% | SD | CV% | SD | CV% | SD | CV% |
| 1 | 0.44 | 0.03 | 6.7 | 0.04 | 8.1 | 0.01 | 2.9 | <0.01 | 0.01 | 0.04 | 9.1 |
| 2 | 4.5 | 0.18 | 3.8 | 0.27 | 5.5 | 0.14 | 3.2 | 0.03 | 0.76 | 0.30 | 6.6 |
| 3 | 13 | 0.73 | 5.1 | 0.95 | 6.6 | 0.16 | 1.2 | <0.01 | <0.01 | 0.96 | 7.2 |
| 4 | 32 | 1.6 | 4.8 | 2.3 | 6.8 | 1.2 | 4.0 | 0.36 | 1.2 | 2.6 | 8.3 |

{14}------------------------------------------------

Image /page/14/Picture/1 description: The image shows the logo for PerkinElmer. The logo consists of a blue abstract shape resembling a stylized "P" with a forward-pointing arrow, positioned above the company name "PerkinElmer" in a bold, sans-serif font. Below the company name, the tagline "For the Better" is written in a smaller, italicized font.

| C4 | | | | | | | | | | | |
|--------|-------------------------|---------------|-----|------------|-----|-------------|------|------------------------|-------|--------------------|-----|
| Sample | Total<br>mean<br>μmol/L | Repeatability | | Within-Lab | | Between-lot | | Between-<br>instrument | | Total<br>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 | 0.01 | 0.01 | 3 |
| 2 | 0.57 | 0.03 | 4.9 | 0.03 | 5.5 | 0.02 | 2.9 | <0.01 | 0.78 | 0.04 | 6 |
| 3 | 1.8 | 0.09 | 4.8 | 0.09 | 4.8 | 0.01 | 0.31 | <0.01 | <0.01 | 0.09 | 4 |
| 4 | 4.2 | 0.18 | 4.1 | 0.20 | 4.6 | 0.08 | 2.0 | 0.04 | 0.93 | 0.22 | 5 |

# CE

| Sample | Total<br>mean<br>μmol/L | Repeatability | | Within-Lab | | Between-lot | | Between-<br>instrument | | Total<br>Variation | |
|--------|-------------------------|---------------|-----|------------|-----|-------------|-----|------------------------|-------|--------------------|-----|
| | | SD | CV% | SD | CV% | SD | CV% | SD | CV% | SD | CV% |
| 1 | 0.04 | 0.01 | 19 | 0.01 | 19 | <0.01 | 2.6 | <0.01 | 0.01 | 0.01 | 17 |
| 2 | 1.0 | 0.04 | 4.2 | 0.06 | 6.3 | 0.03 | 2.8 | <0.01 | <0.01 | 0.07 | 6.7 |
| 3 | 3.6 | 0.17 | 4.9 | 0.18 | 5.2 | 0.04 | 1.1 | <0.01 | <0.01 | 0.19 | 5.2 |
| 4 | 8.9 | 0.37 | 4.2 | 0.43 | 4.9 | 0.31 | 3.6 | <0.01 | <0.01 | 0.53 | 6.0 |

# C5DC\C6OH

| Sample | Total<br>mean<br>μmol/L | Repeatability | | Within-Lab | | Between-lot | | Between-<br>instrument | | Total<br>Variation | |
|--------|-------------------------|---------------|-----|------------|-----|-------------|-----|------------------------|-------|--------------------|-----|
| | | SD | CV% | SD | CV% | SD | CV% | SD | CV% | SD | CV% |
| 1 | 0.03 | 0.01 | 17 | 0.01 | 23 | <0.01 | 9.2 | <0.01 | 5.5 | 0.01 | 25 |
| 2 | 0.44 | 0.03 | 7.5 | 0.03 | 7.9 | 0.01 | 3.1 | 0.01 | 1.5 | 0.04 | 8.6 |
| 3 | 1.5 | 0.09 | 5.9 | 0.10 | 6.2 | 0.02 | 1.1 | <0.01 | <0.01 | 0.10 | 6.3 |
| 4 | 3.8 | 0.22 | 5.7 | 0.24 | 6.2 | 0.07 | 1.9 | 0.07 | 1.7 | 0.26 | 6.7 |

# CE

| Sample | Total<br>mean<br>μmol/L | Repeatability | | Within-Lab | | Between-lot | | Between-<br>instrument | | Total<br>Variation | |
|--------|-------------------------|---------------|-----|------------|-----|-------------|-----|------------------------|-------|--------------------|-----|
| | | SD | CV% | SD | CV% | SD | CV% | SD | CV% | SD | CV% |
| 1 | 0.38 | 0.02 | 4.4 | 0.02 | 6.0 | 0.01 | 2.4 | <0.01 | <0.01 | 0.02 | 6.4 |
| 2 | 1.4 | 0.07 | 5.0 | 0.07 | 5.0 | 0.03 | 1.8 | 0.01 | 0.59 | 0.07 | 5.3 |
| 3 | 3.4 | 0.16 | 4.7 | 0.17 | 4.9 | 0.10 | 3.1 | <0.01 | <0.01 | 0.20 | 5.8 |

C8

| Sample | Total<br>mean<br>μmol/L | Repeatability | | Within-Lab | | Between-lot | | Between-<br>instrument | | Total<br>Variation | |
|--------|-------------------------|---------------|-----|------------|-----|-------------|-----|------------------------|-------|--------------------|-----|
| | | SD | CV% | SD | CV% | SD | CV% | SD | CV% | SD | CV% |
| 1 | 2.0 | 0.10 | 4.8 | 0.12 | 6.0 | 0.08 | 3.8 | <0.01 | <0.01 | 0.14 | 7.1 |
| 2 | 7.5 | 0.35 | 4.7 | 0.37 | 4.9 | 0.12 | 1.6 | <0.01 | <0.01 | 0.39 | 5.2 |
| 3 | 18 | 0.86 | 4.6 | 0.99 | 5.3 | 0.64 | 3.6 | 0.10 | 0.55 | 1.2 | 6.4 |
| 4 | 35 | 1.3 | 3.5 | 1.6 | 4.4 | 0.44 | 1.3 | 0.56 | 1.6 | 1.7 | 5.0 |

CV% 11 6.2 4.8 5.2

{15}------------------------------------------------

Image /page/15/Picture/1 description: The image shows the logo for PerkinElmer. The logo consists of a blue stylized "P" above the name "PerkinElmer" in black font. Below the name is the tagline "For the Better" in a smaller, italicized blue font.

# C10

| Sample | Total<br>mean | Repeatability | | Within-Lab | | Between-lot | | Between-<br>instrument | | Total<br>Variation | |
|--------|---------------|---------------|-----|------------|-----|-------------|-----|------------------------|-------|--------------------|-----|
| | μmol/L | SD | CV% | SD | CV% | SD | CV% | SD | CV% | SD | CV% |
| 1 | 0.46 | 0.02 | 5.5 | 0.03 | 6.8 | 0.01 | 2.4 | <0.01 | <0.01 | 0.03 | 7.2 |
| 2 | 1.6 | 0.09 | 5.8 | 0.11 | 7.1 | 0.02 | 1.2 | <0.01 | <0.01 | 0.11 | 7.1 |
| 3 | 3.9 | 0.24 | 6.2 | 0.29 | 7.7 | 0.11 | 2.9 | 0.04 | 1.1 | 0.32 | 8.2 |

# C12

| Sample | Total<br>mean<br>μmol/L | Repeatability | | Within-Lab | | Between-lot | | Between-<br>instrument | | Total<br>Variation | |
|--------|-------------------------|---------------|-----|------------|-----|-------------|------|------------------------|-------|--------------------|-----|
| | | SD | CV% | SD | CV% | SD | CV% | SD | CV% | SD | CV% |
| 1 | 0.49 | 0.02 | 5.1 | 0.03 | 6.4 | 0.02 | 3.4 | <0.01 | <0.01 | 0.04 | 7.2 |
| 2 | 1.8 | 0.10 | 5.8 | 0.10 | 5.8 | 0.01 | 0.76 | <0.01 | <0.01 | 0.10 | 5.8 |
| 3 | 4.3 | 0.25 | 5.8 | 0.26 | 6.0 | 0.16 | 3.7 | <0.01 | <0.01 | 0.30 | 7.0 |

## C14

| Sample | Total<br>mean<br>μmol/L | Repeatability | | Within-Lab | | Between-lot | | Between-<br>instrument | | Total<br>Variation | |
|--------|-------------------------|---------------|-----|------------|-----|-------------|-----|------------------------|-------|--------------------|-----|
| 1 | 0.05 | <0.01 | 9.0 | 0.01 | 10 | <0.01 | 3.5 | <0.01 | 0.02 | 0.01 | 11 |
| 2 | 0.56 | 0.03 | 5.1 | 0.03 | 6.0 | 0.01 | 1.6 | 0.01 | 1.9 | 0.04 | 6.4 |
| 3 | 1.8 | 0.09 | 5.0 | 0.09 | 5.0 | 0.03 | 1.6 | <0.01 | <0.01 | 0.09 | 5.3 |
| 4 | 4.3 | 0.24 | 5.7 | 0.25 | 5.8 | 0.12 | 2.8 | 0.09 | 1.9 | 0.29 | 6.8 |

## C16

| Sample | Total<br>mean<br>μmol/L | Repeatability | | Within-Lab | | Between-lot | | Between-<br>instrument | | Total<br>Variation | |
|--------|-------------------------|---------------|-----|------------|-----|-------------|------|------------------------|-------|--------------------|-----|
| | | SD | CV% | SD | CV% | SD | CV% | SD | CV% | SD | CV% |
| 1 | 0.99 | 0.05 | 5.1 | 0.06 | 5.8 | 0.02 | 2.3 | <0.01 | <0.01 | 0.06 | 6.1 |
| 2 | 3.6 | 0.16 | 4.4 | 0.20 | 5.7 | 0.13 | 3.6 | <0.01 | <0.01 | 0.24 | 6.6 |
| 3 | 11 | 0.52 | 5.0 | 0.66 | 6.4 | 0.07 | 0.62 | <0.01 | <0.01 | 0.67 | 6.3 |
| 4 | 24 | 1.2 | 5.2 | 1.5 | 6.3 | 0.35 | 1.5 | <0.01 | <0.01 | 1.5 | 6.4 |

# C18

| Sample | Total<br>mean<br>μmol/L | Repeatability | | Within-Lab | | Between-lot | | Between-<br>instrument | | Total<br>Variation | |
|--------|-------------------------|---------------|-----|------------|-----|-------------|------|------------------------|-------|--------------------|-----|
| | | SD | CV% | SD | CV% | SD | CV% | SD | CV% | SD | CV% |
| 1 | 0.49 | 0.03 | 5.5 | 0.03 | 5.9 | 0.01 | 2.0 | <0.01 | 0.01 | 0.03 | 6.2 |
| 2 | 1.2 | 0.06 | 4.5 | 0.08 | 6.1 | 0.03 | 2.7 | 0.01 | 1.1 | 0.09 | 6.9 |
| 3 | 2.9 | 0.15 | 4.9 | 0.15 | 5.1 | 0.03 | 1.0 | <0.01 | <0.01 | 0.16 | 5.4 |
| 4 | 6.2 | 0.28 | 4.4 | 0.34 | 5.3 | 0.03 | 0.58 | <0.01 | <0.01 | 0.34 | 5.5 |

{16}------------------------------------------------

Image /page/16/Picture/1 description: The image shows the logo for PerkinElmer. The logo consists of a blue abstract shape above the company name, "PerkinElmer," which is written in a bold, sans-serif font. Below the company name is the tagline "For the Better" in a smaller, italicized font.

| SA | | | | | | | | | | | |
|--------|-------------------------|---------------|-----|------------|-----|-------------|-----|------------------------|-------|--------------------|-----|
| Sample | Total<br>mean<br>μmol/L | Repeatability | | Within-Lab | | Between-lot | | Between-<br>instrument | | Total<br>Variation | |
| 1 | 0.39 | 0.04 | 17 | 0.05 | 19 | 0.01 | 2.1 | <0.01 | 0.02 | 0.05 | 13 |
| 2 | 3.5 | 0.20 | 6.3 | 0.25 | 7.9 | 0.09 | 2.4 | <0.01 | 0.01 | 0.27 | 7.7 |
| 3 | 13 | 0.62 | 5.4 | 1.0 | 8.6 | 0.22 | 1.6 | <0.01 | <0.01 | 1.0 | 8.1 |
| 4 | 35 | 1.3 | 4.0 | 2.6 | 7.8 | 0.77 | 2.2 | 0.01 | 0.04 | 2.7 | 7.8 |
| 5 | 85 | 5.4 | 5.6 | 8.4 | 8.7 | 1.7 | 3.1 | <0.01 | <0.01 | 8.6 | 10 |

### ADO

| Sample | Total<br>mean<br>μmol/L | Repeatability | | Within-Lab | | Between-lot | | Between-<br>instrument | | Total<br>Variation | |
|--------|-------------------------|---------------|-----|------------|-----|-------------|-----|------------------------|-------|--------------------|-----|
| | | SD | CV% | SD | CV% | SD | CV% | SD | CV% | SD | CV% |
| 1 | 0.13 | 0.02 | 19 | 0.02 | 20 | <0.01 | 2.1 | <0.01 | 0.02 | 0.02 | 17 |
| 2 | 1.4 | 0.07 | 5.1 | 0.10 | 6.8 | 0.02 | 1.3 | <0.01 | <0.01 | 0.10 | 7.1 |
| 3 | 5.7 | 0.20 | 3.5 | 0.22 | 3.8 | 0.07 | 1.2 | <0.01 | <0.01 | 0.23 | 4.0 |
| 4 | 15 | 0.50 | 3.2 | 0.58 | 3.7 | 0.23 | 1.6 | 0.04 | 0.28 | 0.62 | 4.1 |
| 5 | 30 | 0.81 | 2.6 | 1.2 | 3.8 | 0.43 | 1.5 | 0.35 | 1.2 | 1.3 | 4.3 |

### C26:0-LPC

| Sample | Total<br>mean<br>μmol/L | Repeatability | | Within-Lab | | Between-lot | | Between-<br>instrument | | Total<br>Variation | |
|--------|-------------------------|---------------|-----|------------|-----|-------------|------|------------------------|-------|--------------------|-----|
| | | SD | CV% | SD | CV% | SD | CV% | SD | CV% | SD | CV% |
| 1 | 0.26 | 0.04 | 16 | 0.06 | 23 | 0.01 | 4.5 | <0.01 | 0.01 | 0.06 | 23 |
| 2 | 0.78 | 0.08 | 10 | 0.10 | 12 | 0.01 | 1.1 | <0.01 | <0.01 | 0.10 | 12 |
| 3 | 1.5 | 0.11 | 7.1 | 0.11 | 7.7 | 0.01 | 0.47 | <0.01 | <0.01 | 0.11 | 7.7 |
| 4 | 3.0 | 0.18 | 5.8 | 0.19 | 6.4 | 0.01 | 0.55 | 0.10 | 3.0 | 0.22 | 7.3 |
| 5 | 5.5 | 0.29 | 5.0 | 0.37 | 6.5 | 0.10 | 2.1 | 0.11 | 1.9 | 0.40 | 7.3 |

{17}------------------------------------------------

Image /page/17/Picture/1 description: The image shows the logo for PerkinElmer. The logo consists of a blue abstract shape resembling a stylized "P" or an arrow pointing to the right, positioned above the company name. The text "PerkinElmer" is written in a bold, sans-serif font, with the "Elmer" portion slightly bolder than "Perkin". Below the company name, the tagline "For the Better" is written in a smaller, italicized font, also in blue.

#### Analytical Sensitivity:

The analytical sensitivity (the lowest measurable analyte concentrations) has been demonstrated using two QSights. Results are summarized in the table below.

Analytical sensitivity limits for the NeoBase 2 Non-derivatized MSMS kit using QSight:

| Analyte | QSight<br>Analytical sensitivity limit (μmol/L) |
|----------------|-------------------------------------------------|
| Ala | 3.66 |
| Arg | 0.64 |
| Asa 1 | 0.16 |
| Cit | 2.63 |
| Gln\Lys | 6.31 |
| Gly | 8.61 |
| Leu\Ile\Pro-OH | 0.40 |
| Met | 1.56 |
| Orn | 1.83 |
| Phe | 0.29 |
| Pro | 0.34 |
| Tyr | 1.84 |
| Val | 0.84 |
| C0 | 0.18 |
| C2 | 0.04 |
| C3 | 0.02 |
| C4 | 0.01 |
| C5 | 0.01 |
| C5DC\C6OH | 0.04 |
| C6 | 0.03 |
| C8 | 0.10 |
| C10 | 0.04 |
| C12 | 0.10 |
| C14 | 0.01 |
| C16 | 0.02 |
| C18 | 0.01 |
| SA | 0.24 |
| ADO | 0.07 |

¹ Asa is measured as a total concentration of Asa and its anhydrides.

{18}------------------------------------------------

Image /page/18/Picture/1 description: The image shows the logo of PerkinElmer, a global company focused on improving human and environmental health. The logo features a stylized blue "P" shape with a triangular cutout, positioned above the company name. Below the name, the tagline "For the Better" is written in a smaller, italicized font, also in blue.

### Linearity:

The linearity was determined in accordance with CLSI document EP06-A using one QSight. The assay is demonstrated to be linear as presented in the table below.

| Analyte | Linear range lower<br>limit (µmol/L) | Linear range upper<br>limit (µmol/L) |
|----------------|--------------------------------------|--------------------------------------|
| Ala | 163 | 1450 |
| Arg | 1.84 | 359 |
| Asa 1 | 0.22 | 67.2 |
| Cit | 9.18 | 1040 |
| Gln\Lys | 42.2 | 2450 |
| Gly | 268 | 2070 |
| Leu\lle\Pro-OH | 57.8 | 1430 |
| Met | 1.66 | 802 |
| Orn | 25.6 | 807 |
| Phe | 20.9 | 1500 |
| Pro | 37.3 | 1240 |
| Tyr | 19.7 | 1270 |
| Val | 51.0 | 1130 |
| C0 | 7.80 | 407 |
| C2 | 3.53 | 147 |
| C3 | 0.41 | 64.2 |
| C4 | 0.05 | 11.3 |
| C5 | 0.04 | 17.8 |
| C5DC\C6OH | 0.04 | 7.30 |
| C6 | 0.03 | 7.99 |
| C8 | 0.10 | 41.2 |
| C10 | 0.04 | 7.24 |
| C12 | 0.10 | 10.3 |
| C14 | 0.05 | 9.45 |
| C16 | 0.84 | 46.7 |
| C18 | 0.48 | 12.8 |
| SA | 0.24 | 88.2 |
| ADO | 0.17 | 41.3 |
| C26:0-LPC | 0.22 | 7.08 |

Linear ranges determined for the NeoBase 2 Non-derivatized MSMS kit using QSight:

4 Asa is measured as a total concentration of Asa and its anhydrides.

{19}------------------------------------------------

Image /page/19/Picture/1 description: The image shows the logo for PerkinElmer. The logo consists of a blue abstract symbol above the company name, "PerkinElmer," which is written in a bold, sans-serif font. Below the company name is the tagline "For the Better" in a smaller, italicized font, also in blue. The abstract symbol appears to be a stylized arrow or chevron shape, with a darker blue vertical bar on the left and a lighter blue, curved shape on the right.

#### Interference:

The NeoBase 2 Non-derivatized MSMS kit was evaluated for interference in accordance with CLSI document EP07 using QSight. The substances potentially interfering with the assay and additional 9 substances that are abundant in the DBS sample matrix and have potential for unspecific effects on the test results were further tested. The substances potentially interfering with the assay were added into whole blood. The interference samples included NeoBase 2 Control analytes at two concentrations (below and above typical cut-off range). The NeoBase 2 surrogate analytes, which are not included in NeoBase 2 Controls, were studied at endogenous concentration level. The following substances were found not to interfere with the assay on QSight 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 |
| Lidocaine | 51.2 µmol/L |

In the study, the following interferents were identified:

#### Sarcosine:

Amino acid derivative Sarcosine was found to interfere with the assay by increasing the measured Ala concentration. Sarcosine concentrations above 31.3 umol/L may theoretically cause a false positive screening result. Sarcosine concentration in plasma ranges from 0-625 umol/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, Glu and Leu concentrations. Creatine concentrations above 450 umol/L with Ala, above 900 umol/L with Glu or above 1500 umol/L with Leu may theoretically cause a false positive screening result. The normal expected Creatine level in newborns aged 0-1 months is 107-640 umol/L in whole blood. Therefore, Creatine is unlikely to interfere with Glu and Leu in routine testing. The proposed cut-off level measured with NeoBase 2 assay for Ala is approximately 600 umol/L (e.g. 99th percentile, 627 umol/L). An additional 2619 µmol/L dose of Creatine would be needed to raise the Ala concentration from endogenous level (351

{20}------------------------------------------------

Image /page/20/Picture/1 description: The image shows the logo for PerkinElmer. The logo features a blue stylized "P" above the company name, "PerkinElmer," which is written in a bold, sans-serif font. Below the company name is the tagline "For the Better" in a smaller, italicized font, also in blue.

umol/L) to the cut-off level (600 umol/L).

### 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 750 umol/L may theoretically cause a false positive screening result on Orn. The reference plasma level of L-Asparagine in newborns aged 0-1 months is 29-132 umol/L [4]. Therefore, L-Asparagine is unlikely to interfere with Orn in routine testing.

### L-Lysine:

L-Lysine was found to interfere with the assay by decreasing the measured Arg concentrations and by increasing the measured Gln and Glu concentrations above 1000 µmol/L caused a decrease in the measured Arg by 19%. Newborn screening samples with arginine levels close to the cutoff and with suspected hyperlysinemia should be tested using a method that shows no interference by lysine. 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 umol/L, 376-709 umol/L and 62-620 umol/L, respectively. L-Lysine may theoretically 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. 99th percentile, 1200 μmol/L). An additional 1059 µmol/L dose of L-Lysine would be needed to raise the Gln concentration from endogenous level (435 µmol/L) to the cut-off area (1200 µmol/L). For Glu, L-Lysine concentrations from 1500 umol/L may theoretically 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. When considering the positive rate in neonatal screening, it can be expected that any possible effect of endogenous L-lysine would be incorporated in the users established cut-offs for Gln and Glu (upper population percentiles), and would not therefore cause false positives in routine screening. In hyperlysinemia, the concentration of L-Lysine in blood plasma is relatively high. Plasma L-Lysine levels have been reported to exceed 600 umol/L and can reach up to 2000 µmol/L. When blood specimen is taken from 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 concentrations above 2250 µmol/L may theoretically 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-Methionine sulfone:

Amino acid derivative L-Methionine sulfone was found to interfere with…