Evaluation of Millex^® Syringe Filters for Nitrosamine Impurities Testing in Pharmaceutical Drugs

• Nitrosamine Impurities Testing
  • Regulatory Requirements
  • Analytical Methods
  • Sample Filtration Requirements
• Experimental Method
• Evaluation of Filters for Nitrosamines Analysis
  • Extractables
  • Recovery
• Nitrosamine Impurities Testing and Filter Selection
• Related Products
• Related Applications
• References

Nitrosamine Impurities Testing

Detecting and quantifying nitrosamines in medicines is essential to ensure the safety and quality of pharmaceutical products. Nitrosamine chemical impurities are introduced into synthetic processes via multiple and difficult-to-predict routes and are suspected to have carcinogenic and genotoxic properties, posing a risk to patient health.^1-4 They have been detected in drug substances and excipients, leading to many recalls since 2018, including valsartan.^5,6

Regulatory Requirements for Nitrosamines Analysis

Pharmaceutical regulatory agencies and organizations such as the U.S. Food and Drug Administration (FDA), European Medicines Agency (EMA), and International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) have provided guidelines and requirements regarding nitrosamines in pharmaceutical products. A summary of their key points is listed in Table 1.

Table 1. Guidelines and requirements by select pharmaceutical regulatory agencies and organizations that address nitrosamine impurities in pharmaceutical products.

Agency	Nitrosamine Impurities Guideline(s)
U.S. FDA7	Provided guidance with recommended steps that manufacturers of APIs and drug products should take to detect and prevent unacceptable levels of nitrosamine impurities in pharmaceutical products. Expects pharmaceutical companies to identify potential sources of nitrosamine impurities, assess the risk, and take appropriate measures to prevent contamination. Recommends analytical methods such as mass spectrometry and other suitable techniques for analyzing nitrosamines in drug products.
EMA8	Published guidelines and recommendations for the prevention, detection, and risk mitigation of nitrosamine impurities in human medicinal products. Provides thresholds and limits for specific nitrosamine impurities in various drug classes. Manufacturers are required to perform risk assessments, conduct testing, and report any findings of nitrosamine impurities to regulatory authorities.
Health Canada9	Require manufacturers to assess and mitigate the presence of nitrosamines in products, including conducting risk assessments and implementing appropriate preventive measures.
Pharmaceuticals and Medical Devices Agency (PMDA) – Japan10	Issued guidelines on the detection and control of nitrosamine impurities in drug products. The guidelines provide recommendations for risk assessments, testing methods, and strategies for control and prevention.
Health Science Authority (HSA) – Singapore11	Required drug product registrants to conduct risk assessments of their products for nitrosamine impurities. Developed test methods for the identification and determination of nitrosamine impurities in medicines.
Taiwan FDA12	Notified license holders to proactively assess and inspect the risk of nitrosamines impurities in their drug products. Developed recommended test methods for nitrosamine impurities testing.
ICH4	Classified nitrosamines as Class 1 impurities, “known mutagenic carcinogens”.  Issued the guideline M7, which focuses on the assessment and control of DNA-reactive (mutagenic) impurities in pharmaceuticals to limit potential carcinogenic risk. Aims to harmonize existing methods from regulatory agencies and pharmacopoeias across the globe.

These regulatory authorities are actively monitoring and updating their guidelines regarding nitrosamine impurities to ensure public health and safety. It is important for pharmaceutical manufacturers to adhere to these guidelines and requirements while evaluating and mitigating the risk of nitrosamine contamination in medicines.

Analytical Methods for Nitrosamines Analysis

Global regulatory agencies such as EMA, U.S. FDA, and others have emphasized the importance of robust analytical methods for nitrosamine analysis in pharmaceuticals. LC-MS/MS is a commonly recommended analytical method for nitrosamine analysis across regulatory agencies. The combination of high-performance liquid chromatography (HPLC) separation with tandem mass spectrometry detection (MS/MS) enables high sensitivity, selectivity, and accuracy. LC-MS/MS is capable of detecting and quantifying trace levels of the various nitrosamines in complex sample matrices, making it suitable for routine analysis and adherence to regulatory limits. USP <1469> Procedure 3 uses LC-MS/MS for the quantitation of nitrosamines.¹³ The U.S. FDA, European Pharmacopeia,¹⁴ the Health Sciences Agency (HSA) of Singapore,¹⁵ and Taiwan Food and Drug Administration¹⁶ have also published LC-MS/MS methods for determination of nitrosamines in ranitidine and other medicines.

HPLC coupled with High-Resolution Mass Spectrometry (HPLC-HRMS or LC-HRMS) is another key technique for nitrosamine analysis. The selectivity of this method has allowed for the differentiation of nitrosamine-like impurities from actual nitrosamines, ensuring the reliability of test results. The FDA has developed and validated LC-HRMS methods,¹⁷ and USP <1469> Procedure 1 outlines this method as well.⁷

HPLC with UV/Vis detection is also used for nitrosamine analysis. While it may have lower sensitivity compared to LC-MS/MS, it can be used as a quick method for the analysis of raw materials, solvents, or excipients. The Agence Nationale de Sécurité du Médicament et des Produits de Santé (ANSM) has two methods that are based on HPLC-UV.^18,19

Gas chromatography-tandem mass spectrometry (GC-MS/MS) is another commonly employed method for nitrosamine analysis. It offers excellent sensitivity and specificity for nitrosamine detection and is particularly useful for volatile nitrosamines. USP <1469>,¹⁰ Taiwan FDA,¹³ and Swissmedic²⁰ utilize GC-MS/MS methods for nitrosamines testing, and the Health Sciences Agency (HSA) of Singapore published a method based on high-resolution accurate mass gas chromatography-mass spectroscopy (HRAM-GCMS).²¹

Sample Filtration Requirements for Nitrosamines Analysis

Most of the methods mentioned above require filtration of the samples before injection. Filtration is the simplest way to remove particles in samples and mobile phases prior to injection, which could significantly affect the performance of an HPLC instrument. Particulates are very common in drug products such as valsartan, arising from the process of dissolving the formulation. The pore size rating of a membrane filter dictates the extent to which it can retain particulates in a sample; 0.45 µm is common in HPLC methods. When using columns packed with small particles (e.g., sub-2 µm particles) and when using UHPLC, a 0.2 μm filter is recommended. It is important to note that membrane filters with the same pore size retaining do not always have the same retention efficiency, as demonstrated in a previous study.²²

Table 2 lists the filtration steps in specific methods published by regulatory agencies across the globe. For nitrosamines analytical methods, syringe filters are the preferred format. Two important attributes of syringe filters that come into play are the membrane filter material and the pore size. PVDF and PTFE are the most common membrane filter materials called out in published analytical methods; nylon and hydrophilic polypropylene (e.g., GHP membrane) are also mentioned in some cases (Table 2). The pore sizes in the methods are 0.2, 0.22 and 0.45 μm.

Table 2. Filtration requirements in select analytical methods for nitrosamine impurities testing published by agencies across the globe.

Agency	Method	Filtration
U.S. FDA	LC-ESI-HRMS Method for the Determination of Nitrosamine Impurities in Metformin Drug Substance and Drug Product23	“Filter the supernatant using a 0.22 µm PVDF syringe filter”
	LC-HRMS Method for the Determination of Six Nitrosamine Impurities in ARB Drugs24	“Filter the supernatant using a 0.22 µm PVDF syringe filter”
USP10	<1469> Nitrosamine Impurities
	Procedure 1	“Pass the solution through a suitable filter of 0.2-μm pore size.”
	Procedure 3	“Filter into a vial using a hydrophilic polytetrafluoroethylene (PTFE) filter of 0.45-μm pore size”
	Procedure 4	“…0.45-μm nylon filter. Filter 1 mL of sample extract”
European Pharmacopeia	2.5.42. N-Nitrosamines in Active Substances
	Procedure A	“Filter the supernatant through a membrane filter (nominal pore size 0.20 µm)”
	Procedure B Sample Preparation 2	“If necessary, filter the supernatant through a membrane filter (nominal pore size 0.45 µm)”
Agence Nationale de Sécurité du Médicament et des Produits de Santé (ANSM)	Determination of NDMA and NDEA in SARTAN drug substances by HPLC/UV12	“Filter an aliquot … through a 0.45 µm GHP membrane”
	Determination of NDMA in valsartan active substances and finished products by HPLC/UV13	“Filter an aliquot … through a 0.45 μm PVDF membrane”
Health Sciences Agency (HSA) of Singapore	Test method (by LC-MS/MS) for determination of ·   six nitrosamine impurities in western medicines25 ·   N-Nitrosodimethylamine (NDMA) in metformin and ranitidine products26 ·   1-Methyl-4-Nitrosopiperazine (MNP) in rifampicin27 ·   N-Nitroso Salbutamol in salbutamol products28	“Membrane syringe filter (PTFE 0.2 µm)”
	Test method for determination of N-Nitrosodimethylamine (NDMA) in metformin products by HRAM-GCMS15	“Membrane filter (PTFE 0.2 µm)”
Taiwan Food and Drug Administration	Method of Test for Nitrosamines in Medicines - Multiple Analysis (LC-MS/MS Method)29	“Membrane filter: 0.22 μm, PVDF”
	Method of Test for Nitrosamines in Medicines - Multiple Analysis (GC-MS/MS Method)30	“Membrane filter: 0.22 μm, PVDF”

Experimental Methods

A previously validated USP <1469> Procedure 3 method was used in this study.³¹ Part 1 is a study on nitrosamine impurities extractables of syringe filter devices and Part 2 is a recovery study using spiked valsartan. The LC-MS/MS conditions are shown in Table 3 and MRM transitions are shown in Table 4.

Table 3. LC-MS/MS conditions

Instrument:	Agilent 1290 Infinity II instrument coupled to an Agilent 6495C triple quadrupole mass spectrometer
Columns:	Ascentis® Express C18 Column, 2.7 µm, 15 cm x 2.3 mm
Mobile phase:	[A] water + 0.1% formic acid; [B] methanol + 0.1% formic acid
Gradient:	Time (min)			A%	B%
			Initial	97.0	3.0
			1.5	97.0	3.0
			4.0	50.0	50.0
			7.0	25.0	75.0
			8.1	15.0	85.0
			9.2	5.0	95.0
			12.0	5.0	95.0
			12.1	97.0	3.0
Flow rate:	0.50 mL/min
Pressure:	Initial pressure: 320 bar Max. pressure: 460 bar
Column temp.:	60 °C
Detector:	MS, APCI (+), MRM (see Table 4 for details)
Injection:	20 µL
Sample(s):	See text

Table 4. MRM transitions for the six nitrosamine impurities

Peak	Abbreviation	Name	MRM	Collision energy (V)
1	NDMA	Nitrosodimethylamine	75.1->43.3	16
2	NMBA	Nitrosomethylphenylamine	147.1->117.1	4
3	NDEA	Nitrosodiethylamine	103.1->75.1	8
4	NEIPA	Nitrosoethylisopropylamine	117.1->75.1	8
5	NDIPA	Nitrosodiisopropylamine	131.1->43.3	12
6	NDBA	Nitrosodibutylamine	159.2->57.1	12

Syringe Filters Tested. Three devices for each of two lots of (1) Millex^® PVDF 0.22 µm and (2) Millex^® PTFE 0.2 µm and three devices for each of one lot of (3) Supplier P PVDF 0.2 µm, (4) Supplier C H-PTFE 0.2 µm, (5) Supplier M PVDF 0.2 µm, and (6) Supplier M PTFE 0.2 µm were tested.

Part 1 - Extractables. To determine whether there were baseline levels of nitrosamine impurities extractables in syringe filters, a sample of diluent only (0.1% v/v formic acid in water) was spiked with four isotopically labeled internal standards (IS) according to USP <1469> [10 µg/mL for NDMA-d₆ and NMBA-d₃ and 1 µg/mL for NDEA-d₁₀/NDBA-d₁₈]. Samples were vortexed, centrifuged (10,000 rpm for 10 min), and the supernatant filtered using 13 mm syringe filters. The filtrate was analyzed for six nitrosamine compounds using LC-MS/MS. Concentrations were determined using an external calibration curve from 1.33 - 90 ng/mL (NDMA, NMBA, NEIPA, NDIPA and NDBA) and 0.66-69.4 ng/mL (NDEA).

Part 2 - Recovery. 40 mg of valsartan dura (80 mg dose) was pulverized, diluted with 1% formic acid in water, and spiked at low- (L2) concentration according to USP <1469>. The sample was then centrifuged, filtered, and analyzed by LC-MS/MS. 

Evaluation of Filters for Nitrosamines Analysis

Filtration of samples prior to sample injection is part of many LC-MS and GC-MS based methods for nitrosamine impurities testing. Ensuring the absence of extractables in filtration devices is critical for maintaining the accuracy and consistency of data in these methods. Additionally, it is essential to conduct recovery studies, as certain molecules have the potential to bind to the components of a syringe filter and thus may affect the quality of the data generated.

A validated method based on USP <1469> Procedure 3 was used to evaluate the syringe filters.²⁷ This USP method describes the use of LC-MS/MS for the quantitation of NDMA, NDEA, NDIPA, NEIPA, NMBA, and NDBA in selected sartans (valsartan, losartan potassium, olmesartan medoxomil, candesartan cilexetil, and telmisartan). The last part of the “Sample solution” subsection of the method requires filtration “using hydrophilic polytetrafluoroethylene (PTFE) filter of 0.45-μm pore size” after centrifugation.¹¹

Part 1 – Extractables

The filtrate from the syringe filtration step was analyzed for nitrosamine extractables using an external calibration curve from 1.33-90 ng/mL (NDMA, NMBA, NEIPA, NDIPA, and NDBA) and 0.66-69.4 ng/mL (NDEA). Figure 1 is an example of MRM chromatogram of these compounds.

Figure 1. MRM chromatogram of the nitrosamine standard solutions. (10 ng/mL for NDMA, NMBA, NEIPA, NDIPA, and NDBA and 6.6 ng/mL NDEA)

The limit of quantification (LOQ) is defined as the concentration that gives a S/N ratio of 10. The lowest concentration of the calibration curves for each nitrosamine impurity analyzed had S/N >10. No nitrosamine was detected at or above this value in any of the syringe filters tested (Table 5).

Table 5. Results of nitrosamine extractables analysis in various syringe filter devices using USP <1469> Procedure 3.

Compound	Lowest Concentration on Calibration Curve (ng/mL)	S/N	Limit of quantification (LOQ)
			Syringe Filters with PVDF Membrane			Syringe Filters with PTFE Membrane
			Millex® syringe filters	Vendor P	Vendor M	Millex® syringe filters	Vendor C	Vendor M
NDMA	1.33	>10	<1.33	<1.33	<1.33	<1.33	<1.33	<1.33
NMBA	1.33	>10	<1.33	<1.33	<1.33	<1.33	<1.33	<1.33
NDEA	0.66	>10	<0.66	<0.66	<0.66	<0.66	<0.66	<0.66
NEIPA	1.33	>10	<1.33	<1.33	<1.33	<1.33	<1.33	<1.33
NDIPA	1.33	>10	<1.33	<1.33	<1.33	<1.33	<1.33	<1.33
NDBA	1.33	>10	<1.33	<1.33	<1.33	<1.33	<1.33	<1.33

ABBREVIATIONS: S/N = signal-to-noise ratio; PVDF = hydrophilic poly(vinylidene) fluoride; PTFE = hydrophilic polytetrafluorethylene; LOQ = limit of quantitation; ND = not detected

Ensuring that filtration devices do not have chemical extractables that could negatively impact data quality is critical, especially with methods that use highly sensitive instrumentation. Extractables should be avoided because they can co-elute with analyte peaks or show up as unexpected peaks in downstream analyses, making data interpretation more challenging. Sources of extractables could be from shedding of filter, residual chemicals from the manufacturing process, or by secondary chemistries washed off the filter. In the case of specific analytes such as nitrosamine compounds, extractables could be inadvertently formulated during the process of membrane casting.

Part 2 - Recovery in Valsartan

The recovery study was performed by spiking the valsartan sample with reference standards at low- concentration level (L2). The prepared sample solutions were analyzed by LC-MS/MS, using an external calibration curve to calculate the individual analyte concentration. The ratio of internal standard signal versus analyte signal (e.g., signal for NDMA-D₆ / signal for NDMA) was determined in the sample solution and in the external calibration solutions.

Table 6 shows the average recovery for the PVDF and PTFE syringe filters tested. All compound recoveries were within an acceptable range of 70-130%³¹ for all devices and materials tested with some minor differences evident across compounds and filter material. For example, NDBA demonstrated comparatively low recoveries (still within the acceptable range), potentially due to its hydrophobic interactions with filter media or ingredients in the drug product. Both Millex^® hydrophilic PVDF and PTFE syringe filters showed consistent lot-to-lot recoveries.

Table 6. Average percent recovery of six nitrosamine compounds. Values represent the mean ± standard deviation of n=3 devices tested per lot.

Compound	Spike Concentration (ng/mL)	Syringe Filters with PVDF Membrane			Syringe Filters with PTFE Membrane
		Millex®	Vendor P	Vendor M	Millex®	Vendor C	Vendor M
NDMA	2.0	95.6 ± 2.5	95.6 ± 4.0	91.2 ± 1.2	92.7 ± 0.3	94.6 ± 1.0	94.2 ± 1.2
NMBA	2.0	101.1 ± 1.5	99.9 ± 1.6	97.8 ± 0.2	98.6 ± 2.0	97.5 ± 2.3	101.4 ± 0.2
NDEA	0.88	101.4 ± 2.6	101.9 ± 1.2	99.0 ± 3.4	102.4 ± 1.9	100.2 ± 1.9	98.6 ± 1.6
NEIPA	2.0	95.6 ± 1.1	94.6 ± 3.7	92.1 ± 1.1	99.5 ± 3.1	94.6 ± 2.7	93.1 ± 1.3
NDIPA	2.0	92.4 ± 3.8	94.8 ± 3.7	91.6 ± 2.5	99.5 ± 0.6	98.1 ± 0.2	99.2 ± 1.5
NDBA	2.0	83.6 ± 1.8	85.4 ± 5.9	83.6 ± 3.9	82.1 ± 0.9	85.5 ± 1.5	84.2 ± 3.2

Binding of analyte molecules on the filtration device is another factor to consider when choosing syringe filter materials, as binding could lead to poor recoveries. The physico-chemical properties of the filtration device (the membrane filter and housing) and analyte chemistry dictate the extent of binding. Various secondary interactions, such as electrostatic interactions, hydrogen bonding, and hydrophobic interactions, contribute to analyte binding on the membrane filter and housing.³² In this study, the relatively lower recoveries for NDMA using both PVDF and PTFE membrane filters (still within acceptable range) could be the result of hydrophobic interactions between NDMA and the membrane. NDIPA had lower recoveries with the PVDF membranes, indicating that the molecule may interact differently with PTFE versus PVDF polymers. Other polymers with polar functional groups and higher non-specific binding tendencies, such as nylon, would likely demonstrate higher binding, and thus higher losses of analytes. For the most accurate recoveries, it is thus suggested to discard the first mL of filtrate during sample preparation, such as the recommendation given in the U.S. FDA methods for nitrosamines testing.^23,24 This ensures that binding sites are saturated with analyte and thus will not cause additional analyte loss. We have observed this phenomenon for even high binding membrane materials such as nylon.

Nitrosamine Impurities Testing and Filter Selection

All PVDF and PTFE syringe filters tested demonstrated levels of nitrosamine extractables below the limit of quantitation according to USP <1469> Procedure 3. Further, they all gave acceptable recoveries of spiked nitrosamine analytes, showing only minor differences for different compounds. This demonstrates the suitability of these filter media for use in sample preparation for nitrosamine analytical methods.

Related Applications

• Small Molecules Analysis & QC

  Small molecules analysis ensures compliance with pharmacopoeia specifications in drug development.

• Compendial Testing & Regulatory Guidance

  Compendial testing ensures excipients and drug products meet quality standards set by international pharmacopoeia.

References

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2. Cioc RC, Joyce C, Mayr M, Bream RN. 2023. Formation of N-Nitrosamine Drug Substance Related Impurities in Medicines: A Regulatory Perspective on Risk Factors and Mitigation Strategies. Org. Process Res. Dev.. 27(10):1736-1750. https://doi.org/10.1021/acs.oprd.3c00153

3. Li K, Ricker K, Tsai FC, Hsieh CJ, Osborne G, Sun M, Marder ME, Elmore S, Schmitz R, Sandy MS. Estimated Cancer Risks Associated with Nitrosamine Contamination in Commonly Used Medications. IJERPH. 18(18):9465. https://doi.org/10.3390/ijerph18189465

4. International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH). 2023. ICH M7(R2) Guideline on assessment and control of DNA reactive (mutagenic) impurities in pharmaceuticals to limit potential carcinogenic risk.

5. Bharate SS. 2021. Critical Analysis of Drug Product Recalls due to Nitrosamine Impurities. J. Med. Chem.. 64(6):2923-2936. https://doi.org/10.1021/acs.jmedchem.0c02120

6. Maundrell N. 2022. Nitrosamine impurities: from raw materials to final drug product. Bioanalysis. 14(2):63-66. https://doi.org/10.4155/bio-2021-0238

7. FDA. Rigorous Detection of Nitrosamine Contaminants in Metformin Products: Balancing Product Safety and Product Accessibility. . [Internet]. Available from: https://www.fda.gov/drugs/news-events-human-drugs/rigorous-detection-nitrosamine-contaminants-metformin-products-balancing-product-safety-and-product

8. European Medicines Agency (EMA). Nitrosamine impurities. . [Internet]. Available from: https://www.ema.europa.eu/en/human-regulatory-overview/post-authorisation/pharmacovigilance-post-authorisation/referral-procedures-human-medicines/nitrosamine-impurities

9. Health Canada. Nitrosamine impurities in medications: Guidance.

10. Pharmaceuticals and Medical Devices Agency (PMDA). Measures against the risk of nitrosamine contamination in pharmaceuticals. . [Internet]. Available from: https://www.pmda.go.jp/safety/info-services/drugs/0371.html

11. Health Sciences Authority. Nitrosamine impurities in medicines. . [Internet]. Available from: https://www.hsa.gov.sg/therapeutic-products/medicines-quality-and-compliance-monitoring/nitrosamine-impurities-in-medicines

12. Taiwan Food and Drug Administration. 2021 Taiwan Food and Drug Administration Annual Report.

13. USP <1469> Nitrosamine Impurities.

14. European Pharmacopeia. 2.5.42. N-Nitrosamines in active substances.

15. Health Sciences Agency (HSA) of Singapore. Nitrosamine impurities in medicines.. [Internet]. Available from: https://www.canada.ca/en/health-canada/services/drugs-health-products/compliance-enforcement/information-health-product/drugs/nitrosamine-impurities/medications-guidance.html

16. Taiwan Food and Drug Administration. Analytical Methods. . [Internet]. Available from: https://www.fda.gov.tw/eng/siteList.aspx?sid=10360

17. Yang J. 2021. LC-HRMS Based Analytical Platform to Determine Nitrosamines in Pharmaceuticals: Modern Analytical Techniques To Meet Regulatory Needs. 2021 FDA Science Forum.

18. Agence nationale de sécurité du médicament et des produits de santé (ANSM). Method reference: 19A0416-01. Determination of NDMA and NDEA in SARTAN drug substances by HPLC/UV.

19. Agence nationale de sécurité du médicament et des produits de santé (ANSM). Method reference: 18A0399-02. Determination of NDMA in valsartan active substances and finished products by HPLC/UV.

20. Swissmedic. Test method 31_PV_171: Nitrosamines by GC-MS/MS.

21. Health Sciences Agency (HSA) of Singapore. 2021. Test method for determination of N-Nitrosodimethylamine (NDMA) in metformin products by HRAM-GCMS.

22. The Importance of Filtration in Optimizing (U)HPLC and LC-MS Performance.. [Internet]. Available from: /PT/en/technical-documents/technical-article/analytical-chemistry/small-molecule-hplc/hplc-filtration-performance-optimizing#filtration-column

23. FDA. 2020. Liquid Chromatography-Electrospray Ionization-High Resolution Mass Spectrometry (LC-ESI-HRMS) Method for the Determination of Nitrosamine Impurities in Metformin Drug Substance and Drug Product.. [Internet]. Available from: https://www.fda.gov/media/138617/download

24. FDA. 2019. Liquid Chromatography-High Resolution Mass Spectrometry (LC-HRMS) Method for the Determination of Six Nitrosamine Impurities in ARB Drugs.. [Internet]. Available from: https://www.fda.gov/media/125478/download

25. Health Sciences Agency (HSA) of Singapore. 2021. Test method for determination of six nitrosamine impurities in western medicines by LC-MS/MS.

26. Health Sciences Agency (HSA) of Singapore. 2021. Test method for determination of N-Nitrosodimethylamine (NDMA) in metformin and ranitidine products by LC-MS/MS.

27. Health Sciences Agency (HSA) of Singapore. 2021. Test method for determination of 1-Methyl-4-Nitrosopiperazine (MNP) in rifampicin.

28. Health Sciences Agency (HSA) of Singapore. 2021. Test method for determination of N-Nitroso Salbutamol in salbutamol products by LC-MS/MS.

29. Taiwan Food and Drug Administration. 2020. Method of Test for Nitrosamines in Medicines - Multiple Analysis (LC-MS/MS Method).

30. Taiwan Food and Drug Administration. 2020. Method of Test for Nitrosamines in Medicines - Multiple Analysis (GC-MS/MS Method).

31. Muller T, Appelblad P. 2023. Nitrosamine testing using LC-MS methods using USP General Chapter <1469>. Analytix Reporter. 14:15-19.

32. Filter Validation Studies for Pharmaceutical QC – Factors Affecting Analyte Binding. . [Internet]. Available from: https://www.sigmaaldrich.com/technical-documents/technical-article/analytical-chemistry/filtration/millex-filter-analyte-binding