D-778
Limit of Citrinin by LC-MS CURRENT
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1.0 Purpose The purpose of this procedure is to define the method for evaluating the limit of citrinin in raw materials and finished products by LC-MS. 2.0 Scope The method is applicable to all raw materials and finished products with limits in the range of 2 ppb — 40 ppb being tested in the QC laboratory at Ion Labs. 3.0 Responsibility 3.1 It is the responsibility of QC Chemists to follow this procedure. 3.2 It is the responsibility of QC Laboratory Management to ensure that this procedure is being followed. 3.3 It is the responsibility of QC Laboratory Management and/or Analytical Development to keep SOP aligned with current practices. 4.0 Definitions 4.1 LC-MS — Liquid Chromatography with detection by Mass Spectrometry 4.2 HPLC — High Performance Liquid Chromatography 4.3 QC — Quality control 44 CofA — Certificate of analysis 4.5 ACN — Acetonitrile 4.6 H20 — Deionized water 4.7 PVDF - Polyvinylidene fluoride [SOP Standard Operating Procedure SOP No Rev Page Limit of Citrinin by LC-MS D-778 2 of9 5.0 References 5.1 PRTCL-20-0109, Protocol, Validation of an Analytical Method for the Limit of Citrinin by LC-MS 5.2. RPT-21-0022, Report, Estimation of Uncertainty 6.0 Supplies 6.1 Chemicals 6.1.1 Cuitrinin reference standard, 100 ug/mL solution with expanded uncertainty of no more than 0.6%, CAS #518-75-2 (or equivalent) 6.1.2 ACN (LC-MS grade), CAS #75-05-8 (or equivalent) 6.1.3 ACN (HPLC grade), CAS #75-05-8 (or equivalent) 6.1.4 Formic acid (LC-MS grade), CAS #64-18-6 (or equivalent) 6.1.5 Ethanol (ACS/HPLC grade), Catalog #241 HPLC200 (or equivalent) 6.2 Glassware / Disposables 6.2.1 Volumetric glassware as required for standard and sample preparations 6.2.2 Tips for adjustable pipets 6.2.3. 250 uL gas-tight syringe 6.2.4 10-mL plastic syringe and 0.45 um PVDF syringe filter 6.2.5 HPLC vials, 2mL with screw-cap enclosures and septa 6.3 Equipment 6.3.1 Suitable gradient HPLC system consisting of a pump, autosampler, and column oven coupled with Agilent Ultivo mass spectrometer using MassHunter software for instrument control and data processing. 6.3.2. Analytical balance 6.3.3. Wrist action shaker [SOP Standard Operating Procedure SOP No Rev Page Limit of Citrinin by LC-MS D-778 3 3 of9 6.3.4 Adjustable pipets 7.0 Procedure 7.1 Mobile Phase and Diluent Preparation 7.1.1. Mobile Phase A (0.1% formic acid in H2O) 7.1.1.1. Transfer 1000 mL of H20 to a suitable container. 7.1.1.2 Add 1.0 mL of LC-MS grade formic acid. 7.1.1.3. Mix well. 7.1.2 Mobile Phase B (0.1% formic acid in ACN) 7.1.2.1 Transfer 1000 mL of LC-MS grade ACN to a suitable container. 7.1.2.2. Add 1.0 mL of LC-MS grade formic acid. 7.1.2.3 Mix well 7.1.3 Diluent 7.1.3.1 Use HPLC grade ACN. 7.2 Stock Standard Preparation (200 ng/mL) 7.2.1 Citrinin is light sensitive. Prepare all standards and samples in low actinic glassware. 7.2.2 Transfer 200 uL of the reference standard solution to a 100-mL volumetric flask by gas-tight syringe. 7.2.3. Dilute to volume with HPLC grade ACN. 7.3 Spiking Solution Preparation (5 ng/mL) 7.3.1 Transfer 2.5 mL of the Stock Standard to a 100-mL volumetric flask. 7.3.2 Dilute to volume with HPLC grade ACN [SOP Standard Operating Procedure SOP No Rev Page Limit of Citrinin by LC-MS D-778 3 4 of 9 7.4 Sample Preparation 7.4.1 Specific sample testing details are provided in each products profile. If a specific testing details section is not available, then follow preparation procedure as described below, maintaining concentration within the linear range listed above 7.4.2 Sample Weight 7.4.2.1 Based on the limit level, calculate the required sample weight. 7.4.2.2 Example for a sample with 30 ppb limit: Sample Wt (g) = 50 + Limit (ppb) Sample Wt (g) = 50 = 30 ppb Sample Wt (g) = 1.667 g 7.4.3 Unspiked Sample 7.4.3.1 Weigh the required amount of sample (within 0.01g) into a 100-mL low actinic volumetric flask. 7.4.3.2 Add 65 mL of Diluent. 7.4.3.3 Shake on a wrist action shaker for 30 minutes. 7.4.3.4 Dilute to volume with Diluent. 7.4.3.5 Sonicate for 5 minutes. 7.4.3.6 Filter an aliquot using a 0.45 um PVDF membrane discarding the first 2-3 mL before collecting a portion in a HPLC vial for analysis. 7.4.4 Spiked Sample #1 (Spike Concentration = 250 pg/mL) 7.4.4.1 Weigh the required amount of sample (within 0.01g) into a 100-mL low actinic volumetric flask. 7.4.4.2 Add 5.0 mL of Spiking Solution. 7.4.4.3 Add 60 mL of Diluent. 7.4.4.4 Shake on a wrist action shaker for 30 minutes. [SOP Standard Operating Procedure SOP No Rev Page Limit of Citrinin by LC-MS D-778 3 5 of9 7.4.4.5 Dilute to volume with Diluent. 7.4.4.6 Sonicate for 5 minutes. 74.4.7 Filter an aliquot using a 0.45 um PVDF membrane discarding the first 2-3 mL before collecting a portion in a HPLC vial for analysis. 7.4.5 Spiked Sample #2 (Spike Concentration = 500 pg/mL) 7.4.5.1 Weigh the required amount of sample (within 0.01 g) into a 100-mL low actinic volumetric flask. 7.4.5.2 Add 10.0 mL of Spiking Solution. 7.4.5.3 Add 55 mL of Diluent. 7.4.5.4 Shake on a wrist action shaker for 30 minutes. 7.4.5.5 Dilute to volume with Diluent. 7.4.5.6 Sonicate for 5 minutes. 7.4.5.7 Filter an aliquot using a 0.45 um PVDF membrane discarding the first 2-3 mL before collecting a portion in a HPLC vial for analysis. 7.5 HPLC Parameters 7.5.1. Column: Agilent AdvanceBio EC-C18, 2.7 um x 2.1 mm x 100 mm 7.5.2 Column Temperature: 35 °C 7.5.3. Flowrate: 0.25 mL/min 7.5.4 Injection Volume: 5 wL 7.5.5 Run Time: 6.5 minutes 7.5.6 Gradient: Time (min) YA %B 0.0 55 45 3.5 10 90 3.6 55 45 6.5 55 45 [SOP Standard Operating Procedure SOP No Rev Page Limit of Citrinin by LC-MS D-778 3 6 of 9 7.6 MS Parameters 7.6.1 Jon Source: AJS ESI 7.6.2. Gas Temperature: 300 °C 7.6.3. Gas Flow: 12.0 L/min 7.6.4 Nebulizer Pressure: 35 psi 7.6.5 Sheath Gas Temperature: 350 °C 7.6.6 Sheath Gas Flow: 12.0 L/min 7.6.7 Capillary Voltage (Positive Setpoint): 4000 V 7.6.8 Nozzle Voltage (Positive Setpoint): 400 V 7.6.9 Time Segments: Start Time (min) Scan Type 0 MRM 7.6.10 Acquisition Parameters Compound Precursor Product Dwell |Fragmentor . Name ISTD (m/z) MSI1 res (m/z) MS2 res (ms) (V) CE (V) | Polarity Citrinin no 251.2 unit 205.2 unit 400 70 29 is Citrinin no 251.2 unit 233.2 unit 200 70 17 + 7.7 Recommended Sequence 7.7.1. Make at least 2 injections of ACN. 7.7.2 Make a single injection of the Unspiked Sample. 7.7.3. Make a single injection of Spiked Sample #1. 7.7.4 Make a single injection of Spiked Sample #2. 7.7.5 Repeat steps 7.7.2 — 7.7.4, and calculate the average measured concentration from the two replicate measurements. 7.8 System Suitability Requirements 7.8.1 No significant (>5%) interference are present in the diluent injection. [SOP Standard Operating Procedure SOP No Rev Page Limit of Citrinin by LC-MS D-778 3 7 of 9 7.8.2 The coefficient of determination (R’) for a least squares linear regression of the calibration points is no less than 0.99. 7.8.3. The difference in measured concentration between the two replicates is no more than 10% of the limit level. 7.8.4 If results are below LOD, then the system suitability requirements do not apply to the injection that is below LOD. 7.9 Column Wash and Storage 7.9.1. Wash and store the column in Acetonitrile 8.0 Example Calculation 8.1 The calculation may be performed automatically by chromatographic software. 8.2 Plot the data obtained from the unspiked and spiked samples with spike concentration on the x-axis and peak area on the y-axis. 8.3 Perform linear regression of the data to obtain the equation y = mx + b. 8.4 Calculate the concentration of citrinin in the sample: 10#72 x 100 mL xb Citrinin (ppb) = SW Xm b y-intercept of the linear regression SW Sample weight of the unspiked sample in g m slope of the linear regression (mL/pg) 8.5 For results below the detection limit of 2 ppb, the mean of the Unspiked Samples will be reported as “< 2 ppb”. 8.6 For results below the quantitation limit of 6 ppb, the mean of the Unspiked Samples will be reported as “< 6 ppb”. 9.0 Reporting Results 9.1 The expanded uncertainty of the method is 7.1% with a coverage factor of 2. [SOP Standard Operating Procedure SOP No Rev Page Limit of Citrinin by LC-MS D-778 3 8 of 9 9.1.1 Results should include the expanded uncertainty of the method along with the coverage factor in the following format: 9.1.1.1 10 ppb citrinin, U=+ 1 ppb k =2 10.0 Representative Chromatograms * DILUENT Peat aN Standard 500 pg/mL ® | \ i j/ \\ me ° R37189 spike at 30ppb me 190489-14 spike at 20ppb / 11.0 Troubleshooting 11.1 Citrinin can be difficult to wash from the injection system and/or column resulting in a persistent carry-over peak. The carry-over peak can be greatly reduced or eliminated by performing one or more injections of 100 uL of ethanol. [SOP Standard Operating Procedure SOP No Rev Page Limit of Citrinin by LC-MS D-778 3 9 of9 12.0 Revision History | Rev | Date | Description of Changes | CCR # | By | |-----|----------|------------------------|-------|----| | 0 | 06/08/21 | New N/A S. Sassman l 11/09/21 Added 17025:2017 requirements CC-21-0427 J. Sassman Add instruction to follow test details for product specific sample prep, change scope to include anything with a limit in the range 2 | - | - | | 2 | 06/06/23 | — 40 ppb, add option to use alternate reference standard, require CC- filtration of samples instead of settling by gravity, fix calculation so that units are clear. Added statement to say if results are below LOD, then system | 23-0274 | S. Sassman | | 3 | 10/16/25 | suitability requirements do not apply. Updated reporting criteria. CC- Added CAS and Catalog numbers to the supplies list. | 25-0265 | M. Autrey |