Attachment 1 - Testing Instructions.pdf

Attachment 1 – Testing Instructions

Purpose: To determine the sensitivity of LC-triple quad in detecting N-nitrosodimethylamine

(NDMA).

Procedure:

1. Dilute NDMA reference standard solutions into water to the following six concentrations:

0.05 ng/mL, 0.1 ng/mL, 0.2 ng/mL, 0.3 ng/mL, 0.5 ng/mL, and 0.7 ng/mL.

2. Analyze the solutions by triple quad mass spectrometer coupled with LC system.

• A reference LC-MS/MS method is attached. Operation conditions are open for adjustment and optimization. ESI or APCI source can be considered, and APCI may provide better sensitivity.

• Inject each standard solution six consecutive times (injection volume 10 µL). Repeat the cycle for additional three times. The injection sequence is shown below:

NDMA standard solution

No. of injections

Cycle 1

0.05 ng/mL 6

0.1 ng/mL 6

0.2 ng/mL 6

0.3 ng/mL 6

0.5 ng/mL 6

0.7 ng/mL 6

Cycle 2 Repeat Cycle 1

Cycle 3 Repeat Cycle 1

Cycle 4 Repeat Cycle 1

Report must include:

• System identifiers corresponding to proposed system (Make and Model), including other equipment used (columns etc).

• Operational conditions used

• Chromatograms (with and without smoothing) of each injection for one representative cycle;

• %RSD (n = 6) of the peak areas (with and without smoothing) of each standard at each cycle;

• Overall % RSD (n = 24) of the peak areas (with and without smoothing) of each standard;

• Linear regression for one representative cycle.

Attachment: Reference LC-MS/MS method

Reagents

• NDMA Reference Standard

• Formic acid, LC/MS grade (Fisher A117-50 or equivalent)

• Methanol, LC/MS grade (Fisher A456-4 or equivalent)

• Water, LC/MS grade or equivalent

Equipment/Instrument

• HPLC or UHPLC system equipped with temperature-controlled autosampler and column compartment

• LC- Triple Quad system

• HPLC column: ACE Excel C18-AR, 3 µm, 50 x 4.6 mm (MAC-MOD Analytical Inc.

Part No. EXL1190546U) or other C18 column at 50 x 4.6 mm with any particle size.

• HPLC vials

Mobile phase preparation

• Mobile phase A (0.1% formic acid in water): mix formic acid and water at a volume ratio of 1:1000

• Mobile phase B (0.1% formic acid in methanol): mix formic acid and methanol at a volume ratio of 1:1000

Diluent and Blank: Water

Chromatographic Conditions

HPLC Column C18, 50 x 4.6 mm

Column Temp. 30 °C

Flow Rate 0.6 mL/min

Mobile Phase A 0.1% formic acid in water

Mobile Phase B 0.1% formic acid in methanol

Gradient (run time can be adjusted) Time (min) A% B%

0 95 5

5.0 95 5

Injection Volume 10 µL

Autosampler Temp. About 8 °C

Mass spectrometer conditions (please optimize for your system)

• Instrument

Agilent 6420 Triple Quad LC/MS system with APCI source or equivalent.

• Ion Source Settings

Ion Source APCI

Gas Temp. (°C) 325

Vaporizer (°C) 350

Gas flow (L/min) 5

Nebulizer (psi) 40

Capillary (V) 4000

Corona Current (µA) 5

Note: Ion source parameters can be adjusted to achieve the desired sensitivity.

• Scan Settings

Polarity: positive ion;

Scan type: MRM;

Delta EMV(+): 200

Precursor ion

MS1

Res

Product

Ion

MS2

Res Dwell Fragmentor

Collision

Energy

Cell

Accelerator

Voltage

Quantifier 75.1 unit 43.1 unit 200 90 15 5

Qualifier 75.1 unit 58.1 unit 200 90 10 5