RISK MITIGATION OF NITROSAMINES FORMATIONIN DRUG PRODUCTS: Role of Excipients-NEW-UPDATE

How to meet challenges indetermining trace levels ofnitrite in lactose

Ricarda Leister – Head of R&D
MEGGLE Excipients, Wasserburg, Germany

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1. Introduction
In MEGGLE´s whitepaper “Risk Mitigation of Nitrosamines formation in Drug Products: Role of Excipients”
the important issue of Nitrosamine impurities in drug and excipient manufacturing was explained in detailas this topic has become a significant concern for the pharmaceutical industry and health authorities in
the last years. The reason for the update is that MEGGLE invested in the development of a new analyticalmethod to even be able to determine trace levels of Nitrite in Lactose. The new analytical method was
successfully validated in cooperation with the Technical University Munich (TUM).
As known excipients may contribute to the formation of Nitrosamines through precursor substances present in the excipient (e.g. nitrites, amines). Therefore it is an important achievement that with the new
method also trace levels of Nitrite can be determined in MEGGLE´s large Lactose portfolio.

2. Nitrites in MEGGLE Excipients – New Method & Results
MEGGLE has been monitoring nitrite content of Lactose regularly since many years. As nitrate content isalso important for baby food, MEGGLE implemented a method, specifically designed for milk products, andthat is sensitive enough to determine low nitrite content as required for infant milk products (ISO 146733:2007- 05). For MEGGLEs pharma grade Lactose it has been found in the past that nitrite is typically notdetectable in the Lactose powder (below detection limit of 0.2 ppm). Notably all values are lower than themean reported for Lactose by Boetzel et al. (2022).
With the new validated, analytical method the determination even of trace levels of Nitrite in Lactose ispossible. The new method is based Ion chromatography (IC), this is a liquid-solid chromatographic method used to separate and determine ionic solutes. For IC-Analytics different columns as solid phase anddifferent eluent systems as liquid phase can be selected. The exchange and separation of ions depends ontheir charge and affinity towards the applied stationary phase (ion exchange column). The separated ions
are then detected and quantified using conductivity measurement or UV/VIS spectroscopy. The most common used columns are hydroxide-selective anion-exchange columns, carbonate eluent anion-exchange
columns, cation-exchange columns, ion-exclusion columns, amino acid columns and reversed-phase columns. The IC method set-up has been optimized with regards to columns, eluent system, gradient andflow rate as well as the sample preparation to achieve a good resolution for the quantification of Nitrite in
the lactose matrix. Based on the method validation with lactose the Limit of Quantification (LOQ) is 0.03
ppm and the Limit of Detection (LOD) is 0.01 ppm.
After the successful validation of the new method, at least 3 samples from different product groups (sieved,milled, agglomerated, spray dried and anhydrous lactose, as well as milled and sieved inhalation gradelactose) have been analyzed. All results showed a value below 0.01 ppm (table 1).

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Table 1. Nitrites levels in MEGGLE Excipients, measured according the new method. LOQ 0.03 ppm, LOD 0.01 ppm.

Product Group
Sieved Lactose
Milled Lactose
Agglomerated Lactose
Spray Dried Lactose
Anhydrous Lactose
Inhalation GradeLactose sieved
Inhalation GradeLactose milled

Product name

Nitrite Content

Number ofLots Tested

SpheroLac® 100< 0.01 ppm

3< 0.01 ppm

5< 0.01 ppm

3< 0.01 ppm

6< 0.01 ppm

3< 0.01 ppm

3< 0.01 ppm

3GranuLac 200,
GranuLac® 200 US
Tablettose® 70,
Tablettose® 80,
Tablettose® 100
FlowLac® 90,
FlowLac® 100®

DuraLac® H

InhaLac® 120,
InhaLac® 230,
InhaLac® 251
InhaLac® 145,InhaLac® 300,InhaLac® 400

For MEGGLEs pharma grade Lactose portfolio this means that with Nitrite values below 0.01 ppm, they are
not detectable, so basically “nitrite free”. This lowest amount of nitrite also in MEGGLEs direct compressible(DC) lactose grades (agglomerated, spray-dried and anhydrous lactose) makes them a perfect solution tomitigate/reduce risk of nitrosamines formation for drug product manufacturing.

3. IC Application Study for Nitrite Determination
Additionally to the successful validation of the new method with the TUM, MEGGLE also conducted an IC
application study together with the company Thermo Fischer Scientific.
In this study several possible IC set-ups for trace level determination of Nitrite have been tested.
One of the main challenges in this study was the interference from the product matrix (matrix effect).
Lactose contains ions from mineral salt and organic acids in the ppm range. They can interfere with the separation and quantification of the target ions, leading to inaccurate results due to co-elution.
For example, the presence of organic acids such as Lactic acid or Citric acid in the matrix can affect the separation and UV detection at the typically used low wavelength for nitrite (210 nm). Similarly, the presenceof inorganic and organic anions like Chloride, Phosphate, Citrate or Lactate can interfere with conductivity
measurements. To minimize these matrix effects, sample preparation techniques such as in-line dialysis,precipitation or filtration/pre-columns may be used. Selective ion exchange resins and eluents can aid in theseparation of the target ions from the interferences in the matrix (Gapper et al., 200; Jireš, J. & Douša, M.,
2022, Thermo Scientific Application Note 279).

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Several IC system set-ups, like a carbonate system and a gradient hydroxide-system, in combination withconductivity and UV detection have been tested in the Thermo Fischer Scientific application lab. In the test
set-up a matrix elimination system was used.

3.1 Materials and Methods
In this study lactose solutions were prepared by using lactose with a concentration of 50 g/L. The lower
target quantification limit of the method was 20 µg/kg (0.02 ppm) Nitrite related to solid contents, corresponds to 1 µg/l in the test solution. The standard solution contained 1 µg/l Nitrite.
The different columns, eluent and detection systems which were used in this study are listed in table 2.
Table 2. Material and Methods of used IC System. LOQ 0.02-0.05 ppm.

IC System (Dionex™ Integrion™ and Dionex™ Aquion™)
Columns

Eluent System

IonPac™ AS-14A

Isocratic carbonate system

IonPac™ AS11-HC, AS-15,
AS-18, AS-19 and AS-30

Gradient hydroxide system

Detection

Conductivity and UV detection(210 nm)Conductivity and UV detection(210 nm)

These columns are typically used for analysis of inorganic anions e.g. in water analysis.

Low amounts of nitrite could be quantified with the standard solution (see chromatogram 1). However,when it comes to lactose solution it was not possible to quantify Nitrite reliably at this low level due to coelution, neither with UV detection nor with conductivity (see chromatograms 2+3).
Chromatogram 1: standard solution (1 µg/L Nitrite) determined with conductivity

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Chromatogram 2: Lactose solution (50 g/L) determined with conductivity

Chromatogram 3: Lactose solution (50 g/L) overlay with Standard Solution

Therefore, as alternative approach the IC system was combined with a post-column Griess derivatizationand UV detection at 525 nm.
Also in this approach lactose solutions were prepared by using lactose with a concentration of 50 g/L.
The lower target quantification limit of the method was 0.1 µg/l. Nitrite related to solid contents, corresponds to 1 µg/l in the test solution. The standard solution contained 1 µg/l Nitrite.
In this study the Dionex™ Integrion™ with a IonPac™ column AS-22 with NaCl as eluent was combined witha post-column Griess derivatization and UV detection at 525 nm.
With this method 1 µg/l Nitrite could be safely quantified in the lactose solution, no interfering peaks arepresent (see chromatogram 3). The amount of nitrite for the tested GranuLac® 200 sample was estimatedas 2 ppb (0.002 ppm). This is in good agreement with the results of our new validated method (results for
our lactose below detection limit of 0.01ppm).

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Chromatogram 4:
Comparison of the standard (1µg/L Nitrite) with the Lactose solution (50g/L), determined with IC Plus Post-Column Griess Reaction & UV Detection at 525 nm

Standard 1 µg/l

Lactose Solution 50 g/l
Lactose Sample 2 ppb Nitrite (estimated)

The application study has revealed that the determination of nitrite content at trace levels might be challenging and the product matrix needs to be considered.
The Griess reaction is a well-established method commonly used for detecting nitrites in different samples.
This method is based on the selective reaction of nitrite with sulfanilic acid and N-(1-naphthyl) ethylenediamine dihydrochloride under acidic conditions, resulting in a red-violet azo dye that can be measured
using spectrophotometry. By comparing the absorbance values of a sample at 540nm to a predetermined
standard curve, the concentration of nitrite in the sample can be calculated.
Gapper et al. (2004) reported a high-performance ion-exchange methods incorporating on-line postcolumn reduction with either cadmium or vanadium, coupled to derivatization with Griess reagent anddetection at 540 nm, for dairy products and baby foods. This chromatographic separation of nitrate andnitrite, combined with specific post-column conversion to the chromophoric azo derivative, avoids thepotential matrix interference limitations of conventional assays and the inherent disadvantages of otherreported chromatographic detection modes.

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4. Summary & Outlook
In order to minimize the risk of N-Nitrosamine formation in drug products, careful selection of API and excipients in terms of type and amount is crucial. The API manufacturing process and potential impurities orprecursors have to be considered primarily. However, also the drug product manufacturing process itself
can impact the formation of Nitrosamines. Direct compression is the preferred process as it avoids the useof water and heat. Existing formulations can usually be reformulated accordingly.
MEGGLEs specially designed excipients for direct compression can help you to switch from wet-granulation to direct compression, reduce amount of excipients in the formulation, reduce required compression pressure or decrease the amount of disintegrant needed, thus mitigating the risk of N-Nitrosamine
formation.
However, it is important to note that the analytical method used to determine the nitrite content should
be carefully reviewed regarding potential matrix interferences. Moreover, pre-tests on actual blends areadvisable, as not all solid-state interactions driving N-nitrosamine formation in solid dosage formulations
are fully understood yet. Further research is needed to uncover other factors that may affect solid-state reactions, such as local pH gradients, presence of re- active functional groups, counter-ion effects, morphological forms, particle size and surface area of the constituents.
In this paper it was shown that Nitrite determination with IC is possible down to very low levels by using thecorrect method. However this could be a challenge for lactose due to seen co-elution. Therefore it was notpossible to do a reliable quantification of Nitrite at trace level (20 ppm) due to this effect in many different
IC test set-ups (6 different columns were tested). The most sensitive test set-up for quantification of Nitrite
in a lactose solution was achieved by combining IC with post-column Griess derivatization. The estimatedamount of Nitrite for MEGGLEs GranuLac® 200 sample in this test set-up was with 0.002 ppm (2 ppb) extremely low. The reported values for Nitrite in lactose are often significantly higher. With the new validated
method MEGGLE is therefore able to show that their complete lactose portfolio is basically “nitrite free”.

If you would like to learn more about how MEGGLE can help you mitigate the risk associated with
Nitrosamines, please contact us.
We would like to take the opportunity to thank the TUM and Thermo Fischer Scientific for theirengaged, professional and patient collaboration in these studies.

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References
Boetzel, R., Schlingemann, J., Hickert, S., Korn, C., Kocks, G., Luck, B., Blom, G., Harrison, M., François, M., Allain, L. R., Wu, Y. & Bousraf, Y. (2022). A Nitrite Excipient Database: A useful Tool to Support N-Nitrosamine Risk Assessments for Drug Products. Journal of
Pharmaceutical Sciences. https://doi.org/10.1016/j.xphs.2022.04.016
DIN EN ISO 10304-1:2009-07, Wasserbeschaffenheit_- Bestimmung von gelösten Anionen mittels Flüssigkeits-Ionenchroma- tographie_- Teil_1: Bestimmung von Bromid, Chlorid, Fluorid, Nitrat, Nitrit, Phosphat und Sulfat (ISO_10304-1:2007); Deutsche Fassung EN_ISO_10304-1:2009. (2019). Beuth Verlag. https://doi.org/10.31030/1518948
DIN EN ISO 14673-1:2004-05, Milch und Milchprodukte_- Bestimmung des Nitrat- und Nitritgehaltes_- Teil_1: Verfahren mit Cadmiumreduktion und Spektrometrie (ISO_14673-1:2004); Deutsche Fassung EN_ISO_14673-1:2004. (2019). Beuth Verlag. https://doi.
org/10.31030/9540983
DIN EN ISO 14673-2:2004-05, Milch und Milchprodukte_- Bestimmung des Nitrat- und Nitritgehaltes_- Teil_2: Verfahren mit segmentierter Fließanalyse (Routineverfahren) (ISO_14673-2:2004); Deutsche Fassung EN_ISO_14673-2:2004. (2019). Beuth Verlag.
https://doi.org/10.31030/9540984
DIN EN ISO 14673-3:2004-05, Milch und Milchprodukte_- Bestimmung des Nitrat- und Nitritgehaltes_- Teil_3: Verfahren mit
Cadmiumreduktion und Fließinjektionsanalyse mit In-line-Dialyse (Routineverfahren) (ISO_14673-3:2004); Deutsche Fassung EN_ISO_14673-3:2004.(2019). Beuth Verlag. https://doi.org/10.31030/9540985
EMA. Nitrosamine impurities - European Medicines Agency. European Medicines Agency. https://www.ema.europa.eu/en/ human-regulatory/post-authorisation/referral-procedures/Nitrosamine-impurities#guidance-for-marketing-authorisation- holderssection
EMA.Q&A :Questions and answers for marketing authorization holders/applicants on CHMP Opinion for the Article 5(3) of Regulation (EC) No 726/2004 referral on Nitrosamine impurities in human medicinal products EMA/CHMP/409815/2020 Rev.
11. 29 July 2022.
EMA ICH M7 Assessment and control of DNA reactive (mutagenic) impurities in pharmaceuticals to limit potential carcinogenic
risk - Scientific guideline European Medicines Agency. European Medicines Agency. https://www.ema.europa.eu/en/ich-m7- assessment-control-dna-reactive-mutagenic-impurities-pharmaceuticals-limit-potential
Excipients | Lhasa Limited. Lhasa Limited. https://www.lhasalimited.org/initiatives/nitrites.htm
FDA ResearchControl of Nitrosamine Impurities in Human Drugs. U.S. Food and Drug Administration. https://www.fda.gov/ regulatory-information/search-fda-guidance-documents/control-Nitrosamine-impurities-human-drugs
Fritzsche, M., Blom, G., Keitel, J., Goettsche, A., Seegel, M., Leicht, S., Guessregen, B., Hickert, S., Reifenberg, P., Cimelli, A., Baranowski, R., Desmartin, E., Barrau, E., Harrison, M., Bristow, T., O’Neill, N., Kirsch, A., Krueger, P., Saal, C., . . . Schlingemann, J. (2022).
NDMA analytics in metformin products: Comparison of methods and pitfalls. European Journal of Pharmaceutical Sciences, 168,106026. https://doi.org/10.1016/j.ejps.2021.106026
Gapper, L. W., Fong, B., Otter, D., Indyk, H. E. & Woollard, D. C. (2004). Determination of nitrite and nitrate in dairy products byion exchange LC with spectrophotometric detection. International Dairy Journal, 14(10), 881–887. https://doi.org/10.1016/j. idairyj.2004.02.015#Jireš, J. & Douša, M. (2022). Nitrites as precursors of N-nitrosation in pharmaceutical samples – A trace level analysis. Journal of
Pharmaceutical and Biomedical Analysis, 213, 114677. https://doi.org/10.1016/j.jpba.2022.114677

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Thermo Scientific, Application Note 279, Time Savings and Improved Reproducibility of Nitrate and Nitrite Ion Chromatography
Determination in Milk Samples | SelectScience. SelectScience. https://www.selectscience.net/application-notes/time-savings- andimproved-reproducibility-of-nitrate-and-nitrite-ion-chromatography-determination-in-milk-samples/?artID=23548“Wang, Q., Yu, L., Liu, Y., Lin, L. P., Lu, R., Zhu, J., He, L. & Lu, Z. (2017). Methods for the detection and determination of nitrite andnitrate: A review. Talanta, 165, 709–720. https://doi.org/10.1016/j.talanta.2016.12.044

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