Determination of Impurities in Clozapine API using Purospher^® Star RP-18 endcapped HPLC column acc. to Ph. Eur. Monograph

Ajay Kaparwan
Merck Life Science Pvt Ltd, R&D, APAC

Abstract

The clozapine related substances/impurities determination following the European Pharmacopeia (Ph. Eur.) monograph is demonstrated using a Purospher^® Star RP-18 endcapped column. This method showed compliance with the Ph. Eur. system suitability criteria for the clozapine related substances test.

Section Overview

• Introduction
• Experimental
• Results & Discussion
• Conclusion
• Related Products

Introduction

The presence of potential toxic chemicals and impurities in drugs is one of the biggest challenges in the manufacturing of an active pharmaceutical ingredient (API). Therefore, it is important to identify these impurities during the manufacturing process to avoid issues related to the quality, efficacy, and safety of drugs. Screening and quantitation of these impurities in APIs is useful to identify potential problems when evaluating new suppliers, changing manufacturing sites, or during production scale-up. An HPLC-UV method was developed on a Purospher^® STAR RP-18 endcapped column (125 x 4.6 mm, 5 µm) for the simultaneous quantification of the impurities in API for clozapine as per the European Pharmacopoeia method.¹ Beyond what is stated in the European Pharmacopoeia monograph, individual LOD and LOQ limits for the impurities were established by performing an additional calibration for each impurity and clozapine.

Clozapine impurities in the Ph. Eur. monograph are (see structures Figure 1):

• Clozapine Impurity A - 7-chloro-5, 10-dihydro-11H-dibenzo[b,e][1,4]diazepin-11-one
• Clozapine Impurity B - 11,11′-(piperazine-1,4-diyl)bis(8-chloro-5H-dibenzo[b,e][1,4]diazepine)
• Clozapine Impurity C - 8-chloro-11-(piperazin-1-yl)-5H-dibenzo[b,e][1,4]diazepine
• Clozapine Impurity D - 1-[2-[(2-amino-4-chlorophenyl)amino]benzoyl]-4-methylpiperazine

Figure 1. Structures of clozapine and related impurities acc. to European Pharmacopeia (Ph. Eur.).

Experimental

Mobile Phase Preparation

• Solution A: Dissolve 2.04 g of potassium dihydrogen phosphate in 1000 mL of water and adjust the pH to 2.4 + 0.05 with diluted phosphoric acid.

Solution A is mixed with acetonitrile and methanol according to shares described in Table 1 for the mobile phase [A] and [B]. After mixing each mobile phase, sonicate for 20 min and filter through a 0.45 µm membrane filter.

Standard and Sample Preparation

Standards and samples were prepared according to the below procedures:

• Solvent mixture: Methanol and water (80:20 v:v).
• Test Solution: 75 mg of substance to be examined dissolved in Add 80 mL of methanol and swirl gently to dissolve before diluting to 100 mL with water. Mix well and filter through a 0.45 µm membrane filter.
• Reference solution A: 1 mL of Test Solution diluted to 10 mL with solvent mixture. Then 1 mL further diluted to 100 mL with a solvent mixture.
• Reference solution B: Dissolve the contents of a vial of Clozapine for peak identification CRS (containing impurities A, B, C and D) in 1 mL of solvent mixture.
• Stock solution for linearity (15 µg/mL): Weigh and transfer about 1.5 mg of each individual clozapine impurity A, B, C, D, and clozapine standard to 100 mL volumetric flask, respectively, add 80 mL of methanol to each flask and swirl gently to dissolve before diluting to 100 mL with water, then shake and mix well thoroughly.

HPLC-UV Analysis

The standards and samples were analyzed by HPLC-UV at 277 nm using a Purospher^® STAR RP-18 endcapped column (Table 1).

Table 1.HPLC conditions for impurity determination in clozapine acc. to Ph. Eur.

HPLC Parameters
Column:	Purospher® STAR RP-18 endcapped, 5 µm, 125 x 4.6 mm (1.51914)
Mobile phase:	[A] Acetonitrile:methanol:solution A (1:1:8, v:v:v); [B] Acetonitrile:methanol:solution A (4:4:2, v:v:v)
Gradient	Time (min)	%B
	0-4	0
	4-24	0-100
	24-32*	100
Flow rate:	1.2 mL/min
Pressure:	180 bar (2600 psi)
Column temp.:	Ambient
Detector:	UV, 257 nm
Injection volume:	20 µL
Samples:	as described above and in the figures below

Results & Discussion

System Suitability

The system suitability was assessed by comparing the chromatogram acquired with reference solution B (Figure 2) with the chromatogram supplied with Clozapine peak identification CRS (Y0000758; Figure 3), which have to be similar, and the resolution between impurity C and clozapine (>2.5) as indicated in the Ph. Eur. monograph. Both criteria were met. An overview of the suitability criteria and the achieved results is shown in Table 4.

Figure 2. Chromatogram recorded for clozapine reference solution B, prepared in 80% v/v of methanol in water, as per Ph. Eur. monograph for clozapine related substance method.

Figure 3. Example chromatogram provided with clozapine peak identification CRS (Y0000758, batch 3).

Table 2.Peak identification with reference solution B as per Ph. Eur. monograph for clozapine related substance method. Retention time indication Ph. Eur. for clozapine: about 11 min

Peak No.	Name	RT (min)	RRT	RRT in Ph. Eur.	Peak Area	Resolution	Tailing Factor
1	Clozapine Imp C	9.183	0.9	about 0.9	39801	-	1.12
2	Clozapine	10.554	1.0	-	566548	4.80	1.20
3	Clozapine Imp D	13.449	1.3	about 1.1	29520	11.58	1.07
4	Clozapine Imp A	17.306	1.6	about 1.6	174282	18.79	1.06
5	Clozapine Imp B	17.993	1.7	about 1.7	47975	2.95	1.09

Repeatability

The repeatability for the peak area and the retention time was assessed using the reference solution A (Figure 2 & Table 3)

Figure 4. Chromatogram for clozapine reference solution A, prepared in 80% v/v of methanol in water, as per Ph. Eur. monograph for clozapine related substance method (clozapine RT= 10.592 min).

Table 3.Repeatability recorded of Reference solution “A”, prepared in 80% v/v methanol in water

Standard	Peak Area	Retention Time (min)
STD_1	50105	10.592
STD_2	50332	10.591
STD_3	50267	10.593
STD_4	50357	10.594
STD_5	50571	10.591
Mean	50326.4	10.5922
Standard Deviation	168.3324	0.0013
% RSD	0.33	0.01
Ph. Eur. Criteria %RSD	NMT 0.73%	-

Summary of Suitability Criteria

A comparison of system suitability test (SST) criteria as per Ph. Eur monograph for clozapine with the observed value of reference solution A and reference solution B as well as applicability of Ph. Eur 2.2.46 general chapter criteria for % RSD of five replicate injections and tailing factor² are provided in Table 4.

Table 4.Comparison of system suitability test (SST) criteria as per monograph and Ph. Eur 2.2.46 general chapter²

SST Parameters	EP Limits	Observed Value
Resolution between impurity C and clozapine	Minimum 2.5	4.8
Chromatogram obtained with reference solution B	Chromatogram obtained with refence solution B should be similar to chromatogram supplied with clozapine for peak identification CRS.	Chromatogram obtained with refence solution B is similar to chromatogram supplied with clozapine peak identification
Relative retention time with reference to clozapine (retention time about 11 min)	Imp A = about 1.6	Imp A = about 1.63
	Imp B = about 1.7	Imp B = about 1.70
	Imp C = about 0.9	Imp C = about 0.87
	Imp D = about 1.1	Imp D = about 1.27
% RSD of Peak Area reference solution A	NMT 0.73%	0.33%
Tailing factor (as per Ph. Eur 2.2.46)	0.8 – 1.8	Imp A=1.06
		Imp B = 1.09
		Imp C = 1.12
		Imp D = 1.09

Calibration

In addition to what is described in the Ph. Eur. preparation of calibration level solutions were done for each individual impurity and clozapine to assess linearity of calibration and limit of detection and quantification (LOD & LOQ). The calibration levels for each individual impurities were run from 25 % to 150% of the limit mentioned in the Ph. Eur. monograph Table 5.

Table 5.Calibration data for clozapine and 5 impurities

Impurity Name	Calibration Range (µg/mL)	Calibration Equation	R2	LOD (µg/mL)	LOQ (µg/mL)
Clozapine	0.1875 -1.125	y = 66137x - 1527.3	0.9945	0.1	0.29
Clozapine Impurity A	0.1875 - 1.125	y = 218340x + 10169	0.9974	0.07	0.2
Clozapine Impurity B	0.375 - 2.250	y = 33950x - 649.67	0.9953	0.18	0.54
Clozapine Impurity C	0.560 - 3.380	y = 18718x + 2969.6	0.9973	0.2	0.61
Clozapine Impurity D	0.375 - 2.250	y = 18041x + 1937.8	0.9952	0.18	0.54

Measurement of a Test Solution

Figure 5. Chromatogram for clozapine test solution, prepared in 80% v/v of methanol in water, as per EP monograph for Clozapine method for related substances.

Table 6.Data obtained for the Impurity percentages present in the test solution of clozapine, as shown in the chromatogram. (Figure 5)

Compound	% Impurity present in test solution
Clozapine Impurity A	4.48
Clozapine Impurity B	0.51
Clozapine Impurity C	-
Clozapine Impurity D	-

Conclusion

The demonstrated method shows that with the Purospher^® Star RP-18 endcapped, 5 µm, 125 x 4.6 mm column the system suitability criteria for the determination of clozapine related impurities following the Ph. Eur. monograph can be met. The resolution obtained between the peaks of clozapine and clozapine impurity C is 4.8. The relative retention time obtained for the impurities were also comparable with the values listed in the EP monograph.

Although the EP monograph method is used for impurity determination by comparing peak areas, this method here was taken one step ahead beyond the requirements of the Ph. Eur. monograph, by exactly calibrating each individual impurity with single compounds solutions in the concentration range of 25% -150% of the limits mentioned in the monograph. The method showed for the applied concentration ranges a linear behavior with R² values ≥0.995. The determined LODs and LOQs for the HPLC method were between 0.07 - 0.20 mg/mL and 0.20 - 0.61 µg/mL respectively.

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References

1. Clozapine European Pharmacopoeia 11.7. [Internet]. Available from: https://pheur.edqm.eu/app/11-7/content/11-7/1191E.htm

2. Ph. Eur. 2.2.46 general chapter. Chromatographic Separation Techniques. European Pharmacopoeia 11.7. [Internet]. Available from: https://pheur.edqm.eu/app/11-7/content/11-7/20246E.htm