Drug Development and Validation of Elemental Impurities Content in Methotrexate Injection USP 250 mg/10 ml by Inductively Coupled Plasma Mass Spectroscopy (ICP-MS)

Received: 01-Nov-2022, Manuscript No. JDAR-22-82120; Editor assigned: 03-Nov-2022, Pre QC No. JDAR-22-82120 (PQ); Reviewed: 17-Nov-2022, QC No. JDAR-22-82120; Revised: 22-Nov-2022, Manuscript No. JDAR-22-82120 (R); Published: 29-Nov-2022, DOI: 10.4303/JDAR/236210

Introduction

ICP-MS (inductively coupled plasma mass spectrometry) is an analytical technique for measuring elements in biological fluids at trace levels i.e., low and ultra-low quantities. Although some laboratories still employ earlier techniques like atomic absorption and atomic emission, there has been a gradual trend toward ICP-MS, notably in the last decade. The ICP-MS technique has the following advantages over other analysis and that are

• Limits of detection are extremely low

• An extensive linear range

• Possibilities for detecting element isotope composition

• Multi-element character and a high sample throughput

• Allows for more sensitive results

In ICP-MS, Atomic elements are ionized after passing through a plasma source. These ions are then separated based on their mass.

Elemental impurities in a therapeutic product have been linked to potential safety and toxicological hazards, hence proper analysis of elemental impurities is critical to reducing patient exposure. “Any elements that are not supposed to be present in the final formulation of a medicinal product are known as elemental impurities”. The goal of pharmaceutical elemental analysis is to find contaminants that could contaminate the finished product. Risk assessment is now a top responsibility for all pharmaceutical firms to guarantee that all pharmaceutical product components and production techniques comply with requirements. This, however, can be a difficult task for producers, especially when all possible sources of contaminants are taken into account. Excipients, water, the active pharmaceutical substances themselves, as well as container systems and production methods, are examples of such sources. When a possible concern is found, further information is needed, and elemental impurity testing becomes the next obstacle.

Methotrexate, the active ingredient in Methotrexate injectable, USP is an immunosuppressant and a mixture of 4-amino-10-methylfolic acid. This article emphasizes on validation of elemental impurity in Methotrexate Injection [1-4].

Materials and Methods

Different types of elemental standard are used in the validation study, which are Methotrexate injection (USP 250 mg/10 mL), Cadmium, Mercury, Lead, Cobalt, Vanadium, Nickel, Lithium, Antimony, Copper Standard (ICP or Equivalent), chemicals used re Conc. Nitric acid (Suprapur), Conc. Hydrochloric Acid-Fisher or equivalent (Trace metal grade), Purified Water-MilliQ ans Instruments used for ICP-MS are from Thermofisher Scientific.

Determination of elemental impurities ICH Class 1 (Cd, Pb, As, Hg), Class 2A (Co, V, Ni), Class 3 (Li, Sb, Cu) content in Methotrexate injection USP 250 mg/10 mL by ICP-MS

Instrument parameters

Measurement Mode : KED

Cool gas flow rate (L/min) (Argon) : 14

He flow rate (L/min) : 4.34

Oxygen flow rate (L/min) : 0.80

Auxiliary flow (L/min) : 0.80

Nebulizer flow (L/min) : 1.04

Stabilization Time : As per KED

Plasma Power : 1550 W

RF Generator Supply Voltage (V) : 40.23

Peristaltic Pump Speed (rpm) : 40

Spray Chamber Temp (°C) : 2.70

Uptake Time : 60 sec

Wash Time : 60 sec

1 Run : 3 Aspiratio

Note: Instrument parameters may differ based on type and make of instrument. Analysis can be performed with or without auto sampler. Before starting analysis carryout, the performances test (Table 1).

Table 1: Specification limits.

For Different Elements like Cadmium (Cd), Lead (Pb), Arsenic (As), Mercury (Hg), Cobalt (Co), Vanadium (V), Nickel (Ni), Lithium (Li), Antimony (Sb), Copper (Cu) corresponding internal standards are used like Tellurium (Te), Bismuth (Bi), Germanium (Ge), Tellurium (Te), ScanScandium (Sc), Scandium (Sc), Scandium (Sc), Beryllium (Be), Tellurium (Te), Scandium (Sc) are used.

Diluent

Transfer 5.0 mL of Concentrated Nitric acid (67%-69%) solution and 5.0 mL of Hydrochloric acid (34%-37%) into 1000 mL volumetric flask dilute to volume with Purified water.

Preparation of standard stock solution (1000 ppm)

Elements like Cadmium (Cd), Lead (Pb), Arsenic (As), Mercury (Hg), Cobalt (Co), Vanadium (V), Nickel (Ni), Lithium (Li), Antimony (Sb), Copper (Cu) each 250 mL bottle containing 1000 ppm standard stock solution are readily available from market from Merck.

Sample preparation system

Microwave reaction system

Make: Anton Paar or Equivalent

Model: Multiwave 3000 or Equivalent

Program for digestion (Four vessels/Eight vessels and 24 Vessels): (Table 2)

Table 2: Sample preparation system.

Preparation of standard solution A

Transfer element (1000 ppm concentration standard solution each) Cd, Pb, As, Hg, Co, V, Ni, Li, Sb, Cu amount to be taken 0.005 mL, 0.013 mL, 0.038 mL, 0.007 mL, 0.013 mL, 0.025 mL, 0.050 mL, 0.624 mL, 0.225 mL, 0.750 mL to 25 mL volumetric flask volume make up with diluent and mixed well.

Preparation of internal standard solution (1 ppm)

Transfer element (1000 ppm concentration standard solution each) Ge, Be, Sc, Te, Bi amount to be taken 0.050 mL each to 50 mL volumetric flask volume make up with diluent and mixed well.

Preparation of linearity levels

Linearity Level 10%, 30%, 50%, 100%, 150%, 200% was prepared.

For Linearity Level 10%, 30%, 50%, 100%, 150%, 200% transferred volume of standard solution A respectively 0.050 mL, 0.150 mL, 0.250 mL, 0.500 mL, 0.750 mL, 1.000 mL and transferred Internal standard (1 ppm) each 0.500 mL to 50 mL volumetric flask and volume make up with diluent and mixed well.

Preparation of sample/standard blank solution

Transfer 0.250 mL Nitric acid and 0.250 ml HCl, in 50 mL graduated centrifuge tube. Add 0.500 mL of internal standard solution (1 ppm) and dilute to volume up to the mark with purified water.

Preparation of test solution

Transfer 0.500 mL of Sample into a teflon digestion vessel of microwave reaction system, add 0.250 mL Nitric acid and 0.250 ml HCl, close the vessel and carry out digestion. After digestion transfer the content of the teflon vessel into a 50 mL graduated centrifuge tube, ringe the teflon digestion vessel with 10 mL of purified water and transfer the rinsing into same volumetric flask. Add 0.500 mL of internal standard solution (1 ppm) and dilute up to the mark with purified water and mix well [5-7].

Procedure

Aspirate Diluent followed by Linearity level solutions. Further aspirate Diluent, Sample blank, Test solution followed by Linearity level-5 solution as bracketing standard (Tables 3 and 4).

Table 3: System suitability.

Table 4: % Recovery of test solution spiked at 100% level.

Results:

1. System meets the system suitability criteria as specified in method of analysis.

2. % Recovery for each analyte was found within 70.0– 150.0 for 100% level.

System suitability criteria:

1. Correlation coefficient should not be less than 0.99.

2. % Recovery for bracketing standard should be within 70.0-150.0.

Calculations

For Cd, Pb, As, Hg, Co, V, Ni, Li, Sb, Cu

Concentration of Analyst in Linearity Level (ppm):

Concentration of element in Test solution (ppm):

Where,

A: Conc. of element in ppm (Available as that on COA)

V1: Volume of standard stock solution (ready to use) taken in “mL”

V2: Make up volume of standard solution taken in “mL”

V3: Volume of standard solution taken in “mL”

V4: Final volume for linearity level preparation in “mL”

I: Ratio of CPS of analyte and internal standard in Test solution.

C: Constant of the calibration curve

m: Slope of the calibration curve

Note: Concentration of element in Test solution will be reported as per values observes through software.

Validation results

Following parameters were validated.

1. Specificity

2. Limit of Quantitation

3. Linearity and Range

4. Precision

a) System precision

b) Method precision

c) Intermediate precision (Ruggedness)

5. Accuracy

6. Solution Stability

Note: More than one parameters were performed at once with relevant sequence having common system suitability. Diluent sample can be interspersed more than one to avoid carryover from previous sample.

Specificity

Purpose: To demonstrate the ability of the method to assess the analyte unequivocally in presence of components which may be expected to be present.

Prepared Diluent, Linearity Levels, Sample/Standard blank and Test solution as per the Analytical method.

Preparation of test solution spiked at 100% level: Transfer 0.500 mL of Sample into a teflon digestion vessel of microwave reaction system, add 0.250 mL Nitric acid and 0.250 ml HCl, close the vessel and carry out digestion. After digestion transfer the content of the teflon vessel into a 50 mL graduated centrifuge tube, ringe the teflon digestion vessel with 10 mL of purified water and transfer the rinsing into same volumetric flask. Add 0.500 mL of Internal standard solution (1 ppm) and dilute up to the mark with purified water and mix well (Tables 5 and 6).

Table 5: System suitability.

Table 6: Specificity % recovery of test solution spiked at 100% level.

Results:

1. System meets the system suitability criteria as specified in method of analysis.

2. % Recovery for each analyte was found within 70.0– 150.0 for 100% level.

Acceptance criteria:

1. System should meet the system suitability criteria as specified in method of analysis.

2. % Recovery for each analyte should be within 70.0– 150.0 for 100% level.

Limit of quantitation

Purpose: To determine the lowest amount of an analyte in a sample that can be determined with acceptable precision and accuracy under the stated experimental conditions.

Prepared Diluent, Linearity Levels and Sample/Standard blank as per Analytical method.

Preparation of LOQ solution: Considered Linearity Level- 2 as LOQ solution for determination (Tables 7 and 8).

Table 7: LOQ % RSD and concentration.

Table 8: Intensity (cps) of LOQ and standard/sample blank.

Results:

1. System meets the system suitability criteria as specified in method of analysis.

2. % RSD of ratio of CPS of analyte of 6 LOQ solutions were found not more than 20.0.

3. Measured values for Blank was lower than the established Limit of Quantitation (LOQ) for each element.

Acceptance criteria:

1. System should meet the system suitability criteria as specified in method of analysis.

2. % RSD of ratio of CPS of analyte of 6 LOQ solutions should not be more than 20.0.

3. Measure values for Blank should be lower than the established Limit of Quantitation (LOQ) for each element.

Conclusion: % RSD of ratio of CPS of analyte of 6 LOQ solutions for all elements are well within acceptance criteria indicating that the method is precise at LOQ level.

Linearity and range

Purpose: To determine the Linearity and Range of the method.

The linearity of an analytical procedure is its ability (within a given range) to obtain test results which are directly proportional to the concentration (amount) of analyte in the sample. The range of an analytical procedure is the interval between the upper and lower concentration (amounts) of analyte in the sample (including these concentrations) for which it has been demonstrated that the analytical procedure has a suitable level of precision, accuracy and linearity.

Prepared Diluent, Linearity Levels and Sample/Standard blank as per Analytical method (Tables 9 and 10).

Table 9: Correlation coefficient (R²), slope and intercept.

Table 10: % RSD of ratio of CPS at LOQ level and linearity level 6.

Results:

1. System meets the system suitability criteria as specified in method of analysis.

2. Correlation coefficients for each element were found not less than 0.99.

3. % RSD of ratio of CPS of analyte and Internal Standard of 6 LOQ Level and Linearity Level 6 solutions were found not more than 20.0.

Acceptance criteria:

1. System should meet the system suitability criteria as specified in method of analysis.

2. Correlation coefficient for each element should not be less than 0.99.

3. % RSD of ratio of CPS of analyte and Internal Standard of 6 LOQ Level and Linearity Level 6 solutions should not be more than 20.0.

Precision

System precision: Purpose: To demonstrate the repeatability under the same operating conditions over a short period of time. System Precision was demonstrated by injecting the 6 Injections for Linearity Level 4 (100% Level).

Prepared Diluent, Linearity Levels and Sample/Standard blank as per Analytical method (Table 11).

Table 11: % RSD of ratio of CPS at linearity level 4.

Results:

1. System meets the system suitability criteria as specified in method of analysis.

2. % RSD of ratio of analyte of 6 Linearity Level-4 solutions were found not more than 20.0.

Acceptance criteria:

1. System should meet the system suitability criteria as specified in method of analysis.

2. % RSD of ratio of CPS of analyte and Internal Standard of 6 Linearity Level-4 solutions should not be more than 20.0

Method precision

Purpose: To demonstrate the repeatability under the same operating conditions over a short period of time. Precision of an analytical procedure is the degree of agreement among the individual test results when the procedure is applied repeatedly to multiple samplings of a homogenous sample. 6 Test solutions from a single batch was prepared independently and analyzed as per method, also prepare 6 spike solutions to achieve limit level concentration (100% Level).

Prepared Diluent, Linearity Levels and Sample/Standard blank as per Analytical method.

Preparation of spike test solution: Transfer 0.500 mL of Sample into a teflon digestion vessel of microwave reaction system, add 0.250 mL Nitric acid and 0.250 ml HCl, close the vessel and carry out digestion. After digestion transfer the content of the teflon vessel into a 50 mL graduated centrifuge tube, ringe the teflon digestion vessel with 10 mL of purified water and transfer the rinsing into same volumetric flask. added 0.500 mL of Standard solution B and 0.500 mL of Internal standard solution (1 ppm) and dilute up to the mark with purified water and mix well [8-12].

Prepared 6 spiked Test solutions as per above mentioned procedure and labelled as Spike test solution_Prep_1, Spike test solution_Prep_2, Spike test solution_Prep_3, Spike test solution_Prep_4, Spike test solution_Prep_5 and Spike test solution_Prep_6 (Table 12).

Table 12: %RSD of spiked test prepapration at 100% linearity level.

Results:

1. System meets the system suitability criteria as specified in method of analysis.

2. % RSD for each element content for spiked sample were found not more than 20.0.

Acceptance criteria:

1. System should meet the system suitability criteria as specified in method of analysis.

2. % RSD of content value computed for each element for 6 spiked sample should not be more than 20.0.

Intermediate precision

Purpose: To demonstrate the precision within-laboratories variations.

The intermediate precision is the degree of agreement among individual test results when the procedure applied repeatedly to multiple test portions of a homogeneous sample by different analyst on different day using different set of standard solutions.

Prepared Diluent, Linearity Levels and Sample/Standard blank as per Analytical method.

Preparation of spike test solution: Transfer 0.500 mL of Sample into a teflon digestion vessel of microwave reaction system, add 0.250 mL Nitric acid and 0.250 ml HCl, close the vessel and carry out digestion. After digestion transfer the content of the teflon vessel into a 50 mL graduated centrifuge tube, ringe the teflon digestion vessel with 10 mL of purified water and transfer the rinsing into same volumetric flask. added 0.500 mL of Standard solution B and 0.500 mL of Internal standard solution (1 ppm) and dilute up to the mark with purified water and mix well.

Prepared 6 spiked Test solutions as per above mentioned procedure and labeled as Spike test solution_Prep_1, Spike test solution_Prep_2, Spike test solution_Prep_3, Spike test solution_Prep_4, Spike test solution_Prep_5 and Spike test solution_Prep_6 (Table 13).

Table 13: %RSD of spiked test prepapration at 100% level.

Results:

1. System meets the system suitability criteria as specified in method of analysis.

2. % RSD of content value computed for each element for 6 spiked samples were found not more than 20.0.

3. Cumulative % RSD of content value computed for each element from twelve preparations of each precision and intermediate precision were found not more than 25.0.

Acceptance criteria:

1. System should meet the system suitability criteria as specified in method of analysis.

2. % RSD of content value computed for each element for 6 spiked sample should not be more than 20.0.

3. Cumulative % RSD of content value computed for each element from twelve preparations of each precision and intermediate precision should not be more than 25.0.

Accuracy

Purpose: To measure the closeness of the test results obtained by a method to the true value.

Accuracy study was performed at following levels by preparing three spike Test solutions each at LOQ, 50%, 100% and 150% level. Prepared Diluent, Linearity Levels, Sample/Standard blank and Test solution as per Analytical method.

Preparation of accuracy solution at LOQ level: Transfer 0.500 mL of Sample into a teflon digestion vessel of microwave reaction system, add 0.250 mL Nitric acid and 0.250 ml HCl, close the vessel and carry out digestion. After digestion transfer the content of the teflon vessel into a 50 mL graduated centrifuge tube, ringe the teflon digestion vessel with 10 mL of purified water and transfer the rinsing into same volumetric flask. added 0.150 mL of Standard solution B and 0.500 mL of Internal standard solution (1 ppm) and dilute up to the mark with purified water and mix well.

Prepared three Accuracy solutions at LOQ level as per above mentioned procedure and labeled as Accuracy Level LOQ_Prep-1, Accuracy Level LOQ_Prep-2, and Accuracy Level LOQ_Prep-3.

Preparation of accuracy solution at 50% level: Transfer 0.500 mL of Sample into a teflon digestion vessel of microwave reaction system, add 0.250 mL Nitric acid and 0.250 ml HCl, close the vessel and carry out digestion. After digestion transfer the content of the teflon vessel into a 50 mL graduated centrifuge tube, ringe the teflon digestion vessel with 10 mL of purified water and transfer the rinsing into same volumetric flask. added 0.250 mL of Standard solution B and 0.500 mL of Internal standard solution (1 ppm) and dilute up to the mark with purified water and mix well.

Prepared three Accuracy solutions at 50% level as per above mentioned procedure and labeled as Accuracy Level 50%_Prep-1, Accuracy Level 50%_Prep-2, and Accuracy Level 50%_Prep-3.

Preparation of accuracy solution at 100% level: Transfer 0.500 mL of Sample into a teflon digestion vessel of microwave reaction system, add 0.250 mL Nitric acid and 0.250 ml HCl, close the vessel and carry out digestion. After digestion transfer the content of the teflon vessel into a 50 mL graduated centrifuge tube, ringe the teflon digestion vessel with 10 mL of purified water and transfer the rinsing into same volumetric flask. added 0.500 mL of Standard solution B and 0.500 mL of Internal standard solution (1 ppm) and dilute up to the mark with purified water and mix well.

Prepared three Accuracy solutions at 100% level as per above mentioned procedure and labeled as Accuracy Level 100%_Prep-1, Accuracy Level 100%_Prep-2, and Accuracy Level 100%_Prep-3

Preparation of accuracy solution at 150% level: Transfer 0.500 mL of Sample into a teflon digestion vessel of microwave reaction system, add 0.250 mL Nitric acid and 0.250 ml HCl, close the vessel and carry out digestion. After digestion transfer the content of the teflon vessel into a 50 mL graduated centrifuge tube, ringe the teflon digestion vessel with 10 mL of purified water and transfer the rinsing into same volumetric flask. added 0.750 mL of Standard solution B and 0.500 mL of Internal standard solution (1 ppm) and dilute up to the mark with purified water and mix well.

Prepared three Accuracy solutions at 150% level as per above mentioned procedure and labelled as Accuracy Level 150%_Prep-1, Accuracy Level 150%_Prep-2, and Accuracy Level 150%_Prep-3 (Table 14).

Table 14: % Recovery.

Results:

1. System meets the system suitability criteria as specified in method of analysis.

2. Each % Recovery for the analyte were found within 70.0–150.0 for all linearity levels.

Acceptance criteria:

1. System should meet the system suitability criteria as specified in method of analysis.

2. % Recovery for each element should be within 70.0– 150.0 for all acuuracy levels.

Solution stability

Purpose: To demonstrate the stability of the analyte in solution.

Two Test solutions from a single batch (as used in Method precision) was prepared to achieve the concentration of about 100% level and another two test solutions from single batch Keep the samples for 24 hours on bench top at ambient condition. Aspirate the solution along with the linearity. Samples from spiked method Precision can be oooused for solution stability (Table 15).

Table 15: Solution stability.

Result:

1. System meets the system suitability criteria as specified in method of analysis.

2. Overall Cumulative % RSD for each element content at 100% Level obtained from Method Precision and solution stability samples was found not more than 20.0.

Acceptance criteria:

1. System should meet the system suitability criteria as specified in method of analysis.

2. Cumulative % RSD of content value computed for each element from Method Precision and solution stability samples should not be more than 20.0.

Discussion

Specificity

Acceptance Criteria -System should meet the system suitability criteria as specified in method of analysis and % Recovery for each analyte should be within 70.0–150.0 for 100% level.

Result-System meets the system suitability criteria as specified in method of analysis and % Recovery for each analyte was found within 70.0–150.0 for 100% level.

Limit of quantitation

Acceptance Criteria-System should meet the system suitability criteria as specified in method of analysis, % RSD of ratio of CPS of analyte of 6 LOQ solutions should not be more than 20.0 and Measured values for Standard/Sample Blank was lower than the established Limit of Quantitation (LOQ) for each element.

Result-System meets the system suitability criteria as specified in method of analysis, % RSD of ratio of CPS of analyte of 6 LOQ solutions were found not more than 20.0 and Measured values for Standard/Sample Blank was lower than the established Limit of Quantitation (LOQ) for each element.

Linearity and range

Acceptance Criteria-System should meet the system suitability criteria as specified in method of analysis, Correlation coefficient for each element should not be less than 0.99 and % RSD of ratio of CPS of analyte and Internal Standard of 6 LOQ Level and Linearity Level 6 solutions should not be more than 20.0.

Result-System meets the system suitability criteria as specified in method of analysis, Correlation coefficient for each element were found not less than 0.99 and % RSD of ratio of CPS of analyte and Internal Standard of 6 LOQ Level and Linearity Level 6 solutions were found not more than 20.0.

Precision

a. System precision-System should meet the system suitability criteria as specified in method of analysis, % RSD of ratio of analyte of 6 Linearity Level-4 solutions should not be more than 20.0.

Result-System meets the system suitability criteria as specified in method of analysis and % RSD of ratio of analyte of 6 Linearity Level-4 solutions were found not more than 20.0.

b. Method Precision-System should meet the system suitability criteria as specified in method of analysis and % RSD for each element content for spiked sample should be NMT 20.0.

Result-System meets the system suitability criteria as specified in method of analysis and % RSD for each element content for spiked sample were found NMT 20.0.

c. Intermediate Precision-System should meet the system suitability criteria as specified in method of analysis, % RSD for 6 replicate injections of spiked test solution should be NMT 20.0 and Overall Cumulative % RSD for each element content at 100% Level obtained from Method Precision and Intermediate Precision studies should not be more than 25.0.

Result-System meets the system suitability criteria as specified in method of analysis, % RSD for 6 replicate injections of spiked test solution were found NMT 20.0 and Overall Cumulative % RSD for each element content at 100% Level obtained from Method Precision and Intermediate Precision studies were found not more than 25.0.

Accuracy

Acceptance Criteria-System should meet the system suitability criteria as specified in method of analysis and each % Recovery for the analyte should be within 70.0–150.0 for all linearity levels.

Result-System meets the system suitability criteria as specified in method of analysis and each % Recovery for the analyte were found within 70.0–150.0 for all linearity levels.

Solution stability

Acceptance Criteria-System should meet the system suitability criteria as specified in method of analysis and Overall Cumulative % RSD for each element content at 100% Level obtained from Method Precision and solution stability samples should not be more than 20.0.

Result-System meets the system suitability criteria as specified in method of analysis and Overall Cumulative % RSD for each element content at 100% Level obtained from Method Precision and solution stability samples was found not more than 20.0

Conclusion

Based upon the data and results obtained for the analytical method validation, the ICP-MS method used for the determination of elemental impurities in Methotrexate injection USP 250 mg/10 mL specific, and selective. The Table method is linear over the range of 10% to 200%. The method is precise. The ICP-MS method for the evaluation elemental impurities in Methotrexate injection USP 250 mg/10 mL has been validated.

Acknowledgement

None.

Conflict of Interest

Authors have no conflict of interest to declare.

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