HPLC Separation of Nitrosamines with Supel^TM Carbon LC

Eddy Tan, Associate Senior Scientist

R&D APAC Lab Singapore

Abstract

The Supel^TM Carbon LC column is used for the separation of six nitrosamine compounds. This method is a reversed phase gradient separation of the nitrosamines for identification and quantification.

Introduction

Experimental

Results and Discussion

Conclusion

Introduction

Nitrosamines have been known to exist in the environment and within food sources^{1, 2} with awareness further raised in 2018 when many regulatory authorities for medicines became aware of the dangers of N-Nitrosamines as drug contaminants.³ This class of compounds is known to be potent carcinogens⁴ and can be introduced through multiple introduction pathways.⁵ In this application, 2.7 µm porous graphitic carbon particles in Supel^TM Carbon LC column are used to separate six different nitrosamines followed by UV detection.

Experimental

For this study, the following procedures and conditions were used:

Mobile Phase Preparation

• Water + 0.1% trifluoroacetic acid (TFA): Add 100 µL of TFA to 100 mL of water.
• Acetonitrile + 0.1% TFA: Add 100 µL of TFA to 100 mL of acetonitrile, add trifluoroacetic acid

Table 1. HPLC Conditions used for nitrosamine determination

HPLC Parameters
Column:	SupelTM Carbon LC, 2.7 µm, 100 x 3.0 mm I.D. (59993-U)
Mobile phase:	[A] Water + 0.1% TFA, [B] Acetonitrile + 0.1% TFA
Gradient:	Time (min)	% B
	0.00	2.5
	0.50	2.5
	5.00	95.0
	7.00	95.0
	7.01	2.5
	10.00	2.5
Flow Rate	0.5 mL/min
Pressure:	80 – 130 bar
Column temp:	90 oC
Detector:	UV, 230 nm
Injection:	5 µL
Samples:	Standard solution (see below)

Nitrosamine Standard Solutions for Linearity, LOD and LOQ Determination

NMBA, NEIPA, NDIPA, NDBA and NMPA

• For NMBA, NEIPA, NDIPA, NDBA and NMPA (standard concentration ~1000 mg/L), transfer 400 µL into a 5 mL volumetric flask.
• Top-up to mark with mobile phase A (water + 0.1% TFA) and mix well. Sonicate for 10 min. This solution is the main working standard (~80 mg/L).
• Dilute to approximately 5, 10, 20, 40, 60 mg/L with mobile phase A in 5 mL volumetric flasks based on serial dilution. (True concentrations for linearity are based on batch CoA of reference materials used.)

NDEA

• For NDEA (Neat liquid ~99.8 % purity), weigh ~50 mg of NDEA CRM into a 5 mL volumetric flask.
• Top-up to mark with acetonitrile and mix well. Sonicate for 10 min. This solution is the stock solution (~10,000 mg/L).
• Dilute to approximately 5, 10, 20, 40, 60, 80 mg/L with mobile phase A in 5 mL volumetric flasks based on serial dilution. (True concentrations for linearity is based on batch CoA of reference material used.)

Linearity data is obtained from linear regression, LOD and LOQ values are calculated based on USP.

Results & Discussion

The analysis of the standard mix containing six nitrosamine impurities (NDEA, NMBA, NEIPA, NDIPA, NDBA, NMPA) at 20 mg/mL each is shown in Figure 1. The chromatographic data for the analysis of the standard mix is listed in Table 2. All peaks are base line separated.

Figure 1. Separation of six nitrosamines standard mix (~20 mg/L each); blank subtracted chromatogram. 1: NDEA, 2: NMBA, 3: NEIPA, 4: NDIPA, 5: NDBA, 6: NMPA.

Table 2. Nitrosamines retention time, resolution and asymmetry for a standard with ~20 mg/L each compound

Peak no.	Compound	Retention time (min)	*Asymmetry (USP)	*Resolution (USP)
1	N-Nitrosodiethylamine (NDEA)	3.1	1.34	2.92
2	N-Nitroso-N-methyl-4-aminobutyric acid (NMBA)	3.4	1.59	2.36
3	N-Nitrosoethylisopropylamine (NEIPA)	3.6	1.26	5.80
4	N-Nitrosodiisopropylamine (NDIPA)	4.1	1.16	18.95
5	N-Nitrosodibutylamine (NDBA)	5.3	1.19	10.69
6	N-Nitrosomethylphenylamine (NMPA)	6.5	1.54	-
*Asymmetry and resolution values were generated from Chromeleon® 7 CDS software.

Calibration

The calibration data summary and combined limits of detection (LOD) and limits of quantification (LOQ) of the applied method for the six nitrosamines (NDEA, NMBA, NEIPA, NDIPA, NDBA, NMPA) are listed in Table 3. All six nitrosamines exhibited excellent linear regression of more than 0.99. LOD and LOQ of these nitrosamines are found to be in low mg/L range when using UV detection.

Table 3. Calibration data summary and Limits of detection (LOD) and limits of quantification (LOQ) for the six nitrosamines

Peak	Compound	Calibration range (mg/L)	No. of calibrators	R2	LOD (mg/L)	LOQ (mg/L)
1	N-Nitrosodiethylamine (NDEA)	11.03 - 88.24	5	0.9998	1.58	4.79
2	N-Nitroso-N-methyl-4-aminobutyric acid (NMBA)	4.76 - 57.16	5	0.9999	0.73	2.21
3	N-Nitrosoethylisopropylamine (NEIPA)	10.51 - 84.02	5	0.9961	7.18	21.75
4	N-Nitrosodiisopropylamine (NDIPA)	5.00 - 60.00	5	0.9998	1.18	3.56
5	N-Nitrosodibutylamine (NDBA)	4.96 – 59.46	5	1.0000	0.41	1.25
6	N-Nitrosomethylphenylamine (NMPA)	9.48 - 75.79	5	0.9996	2.13	6.46

Conclusion

The Supel^TM Carbon LC column is able to separate six nitrosamines based on reversed phase with good reproducibility for repeated analyses (data not shown here). The resolution and peak asymmetry for the six different nitrosamines are greater than 1.5 and less than 2.0 respectively. LOD and LOQ of the tested nitrosamines are in the low mg/L range and can be further improved with the use of mass spectrometry instrumentation.

References

1. Robles H. 2014. Nitrosamines. Encyclopedia of Toxicology (Third Edition). Academic Press.584-585. https://doi.org/10.1016/b978-0-12-386454-3.00523-6

2. Gushgari AJ, Halden RU. 2018. Critical review of major sources of human exposure to N-nitrosamines. Chemosphere. 210:1124-1136. https://doi.org/10.1016/j.chemosphere.2018.07.098

3. Center for Drug Evaluation, Research. FDA updates and press announcements on angiotensin II receptor blocker (ARB) recalls (valsartan, losartan, and irbesartan) . [Internet]. U.S. Food and Drug Administration. 2023 Aug 23 .[cited 31 Aug 2023]. Available from: https://www.fda.gov/drugs/drug-safety-and-availability/fda-updates-and-press-announcements-angiotensin-ii-receptor-blocker-arb-recalls-valsartan-losartan

4. Information note for nitrosamine impurities. 2019. [Internet]. World Health Organization. Available from: https://www.who.int/news/item/20-11-2019-information-note-nitrosamine-impurities

5. USP General Chapter <1469> Nitrosamine Impurities. https://doi.org/10.31003/uspnf_m15715_02_01