1. INTRODUCTION:

The prevention of cross-contamination of drugs in pharmaceutical production must be avoided, therefore the cleaning of the manufacturing equipment is an important aspect of good manufacturing practice. The process of demonstrating the efficiency of the cleaning procedure is known as cleaning validation. Cleaning validation is required to in the pharmaceutical field to avoid potential clinically significant synergistic interactions between pharmacologically active chemicals. The cleaning validation describes responsibilities, facilities, cleaning strategies, analytical strategies and residue limit justifications. The cleaning validation consists therefore in two separate activities: the first is the development and validation of the cleaning procedure that is used to remove drug residues from the manufacturing equipment surfaces and the second consists in developing and validation the methods used to quantify residuals from surfaces that are used in the manufacturing environment.

To monitor the effectiveness of the cleaning operation, the analytical method should be selective for the substance considered and must be of sufficient sensitivity, because residue concentrations are usually low. According to the FDA, the limit should be based on logical criteria involving the risk associated with residues of the product determined. Calculation of an acceptable residue limit and maximum allowable carryover for active products in production equipment should be based on therapeutic doses, toxicological index and a general limit (10 ppm). According to the food and drug administration (FDA), two different method of sampling are generally admitted for performing a cleaning control: the direct surface sampling, using the swabbing technique and the indirect sampling based on the analysis of solutions used for rinsing the equipement (rinse method)^1-11.

Ketorolac tromethamine is a non-steroidal anti-inflammatory drug (NSAID) in the family of heterocyclic acetic acid derivatives, often used as an analgesic, antipyretic, and anti-inflammatory. It acts by inhibition of prostaglandin synthesis by competitive blocking of the enzyme cyclooxygenase (COX). It is available in oral, intramuscular and topical dosage forms^12-13. Structure of Ketorolac tromethamine is given in fig. 1.

Fig. 1: Ketorolac tromethamine

Numerous methods have been published for quantitative analysis of Ketorolac tromethamine and its impurities or metabolites such Spectrophotometric and Spectrofluorometric^14-15, HPLC^16-20, HPTLC²¹, Capillary electrochromatography²² etc. A literature revealed that no validated cleaning method was available for Ketorolac tromethamine.

Taking these consideration into account that the aim of this study was to develop and validate a simple and sensitive analytical method enabling determination of trace levels of Ketorolac tromethamine residues in production area equipment to confirm the efficiency of the cleaning procedure. The analytical method was validated in the terms of selectivity, linearity, accuracy, precision and limit of detection and limit of quantification and solution stability.

2. MATERIALS AND METHODS:

2.1 Instruments and apparatus

Waters Alliance HPLC system connected with PDA detector and Empower software for data acquisition, XS205 Dual range balance (Make: Mettler Toledo), Bandelin Sonorex sonicator, Heraeus Biofuge Stratos Centrifuge and Stainless steel plates (4 cm × 4 cm) were used during development study. Class A Volumetric flasks, pipettes, beakers, measuring cylinders and centrifuge tubes of Borosil glass were used.

2.2 Chemicals and reagents

Ketorolac tromethamine standard, 1-hydroxy analog and 1-keto analog impurity standards were provided by Dr. Reddy’s Laboratories, Hyderabad, India. Methanol-HPLC grade (SDFCL, India), Glacial acetic acid (Merck, India), Water-HPLC grade and 0.45µ PVDF membrane filters were used.

2.3. Method

2.3.1 Chromatographic parameters

All chromatographic experiments were performed in the isocratic mode. Separation was achieved on Symmetry C18, 250 × 4.6 mm, 5 µ by using Waters Alliance HPLC with PDA detector and controlled by Empower software. Mobile phase was a filtered and degassed mixture of Water, Methanol and Glacial acetic acid in the ratio of 54:45:10 (% V/V). Other parameters such as flow rate of 1.2 ml/minute, detection at 313 nm, column temperature of 40°C, injection volume of 20 µl and run time of 30 minutes were finalized during development. Diluent was used as mixture of water and methanol in the ratio of 55:45 (V/V).

2.3.2 Standard solution preparation

Standard stock solution was prepared by weighing about 20.0 mg of Ketorolac tromethamine standard into 200 ml of volumetric flask, added 100 ml of diluent followed by sonication for 5 minutes in ultrasonic bath to dissolve it. Then cooled at room temperature and made up to volume with diluent and mix (Standard stock of Ketorolac tromethamine: 100 µg/ml). Transferred 5.0 ml of this stock solution into 50 ml of volumetric flask and made up to volume with diluent (Standard solution of Ketorolac tromethamine: 10 µg/ml). The standard stock solution was subsequently diluted with diluent to furnish calibration curve (Linearity) in the range of 0.13 – 30 µgmL^-1.

2.3.3 Resolution solution preparation:

Weighed 0.2 mg of 1-hydroxy analogue and 1.0 mg of 1-Keto analogue into 10 ml of volumetric flaks, dissolved and diluted to volume with diluent (Ketorolac impurity stock solution). Transferred 1.0 ml of impurity stock solution and diluted to 10 ml with Ketorolac tromethamine standard stock solution (Resolution solution).

2.3.4 Sample preparation:

Selected stainless-steel surfaces (4 cm × 4 cm), previously cleaned and dried, were sprayed with 1 ml standard stock solution for positive swab control and then solvent was allowed to evaporate (approximate time was 1 hour). The surfaces were wiped with the first cotton swab soaked with methanol, passing it in various directions to remove the residues from the stainless steel. The other dry cotton swab was used to wipe the wet surfaces. The swabs were placed in a 25-ml screw cap test tube containing 10 ml of diluent. The negative swab control was prepared in the same way as the sample, using swabs, which had not been in contact with the test surface. Subsequently, the tubes were shaked for 10 minutes on rotary shaker followed by sonication for 10 minutes in ultrasonic bath. Squeeze the swabs and filter the solutions through 0.45µm PVDF hydrophilic membrane filter and solutions were analyzed by HPLC.

2.3.5 Swab samples from different locations within the Manufacturing equipment:

Swab samples from different locations within the manufacturing equipment were submitted to the laboratory for analysis of Ketorolac tromethamine residues. These samples were prepared and analyzed as described above.

3. RESULTS AND DISCUSSION:

3.1 Acceptance limits calculation:

The maximum allowable carryover (MACO) is the acceptable maximum allowed concentration of previous substance to next batch. The MACO is determined based on the therapeutic dose, toxicity and generally 10 ppm criteria. Based on determined maximum allowable residual limit in subsequent product, the next step was the determination of the residue limit in terms of the contamination level of active ingredient per surface area of equipment. The total surface area of the equipment in direct contact with the product was accounted for in the calculations. The limit for the previous product in the subsequent product is determined by use of the equation:

MACO_PPm(mg) = MB_next(mg) × 10/1000000

Where, MACOPPm is the acceptable amount transferred to the next product, calculated from the general 10 ppm limit, MB_nextis the minimum batch size for the next product, and 10/1000000 denotes 10 ppm. When the maximum allowable residue limit in the subsequent product has been determined, the next step is to determine the residue limit as the level of active ingredient contamination per unit surface area of the equipment. The equation used for determining the limit per surface area are:

LSA (mg cm^-2) = MACO_PPm(mg)/SA (cm²) and

LSA (mg per 16 cm²) = LSA (mg cm^-2)× S (cm²)

Where, LSA is the limit per unit surface area, calculated on the basis of equipment surface area and the most stringent MACO, SA is the equipment surface area in common between one product and the subsequent product, expressed in cm², and S is the swab area (16 cm²).Based on contact surface area for equipment items included in production of Ketorolac tromethamine Gel, the calculated limit per surface area (LSA) in the case of Ketorolac tromethamine was 100 µg/swabfor a 16 cm² area (appropriate concentration 10 µg/ml.

3.2 Optimization of chromatographic conditions

To find the best chromatographic conditions, wavelength for detection, column and mobile phase were adequately selected. The main objective was to develop an RP-HPLC method enabling determination of Ketorolac tromethamine residues collected by swabs without interference from excipients or impurities present in formulation or active pharmaceutical ingredient.With reference to Ketorolac tromethamine injection USP monograph, different L1 columns such as Hypersil BDS C18 and Zorbax C18 were assessed with a mixture of water, methanol and glacial acetic acid in different proportions as mobile phase for good peak symmetry. But problems such as broad peak shape, co-elution of peaks and increase in the column back pressure were observed. Symmetry C18 (250 × 4.6 mm, 5µm) was preferred to improve the peak symmetry and appropriate retention time. The best separation of Ketorolac and its impurities was achieved with the mobile phase as Water: Methanol: Glacial acetic acid (54:45:1, % v/v) at a flow rate of 1.2 ml/minute and in isocratic mode.

Wavelength for detection was selected as 313.0 nm because Ketorolac tromethamine has a sufficient absorbance at this wavelength and enough to detect ketorolac tromethamine and its impurities at lower concentration.

Other chromatographic parameters such as column temperature of 40°C, injection volume 20 µl and run time of 30 minutes were finalized during development study.

1-hydroxy analog, 1-keto analog and Ketorolac peaks were eluted about 13.0, 17.0 and 21.0 minutes respectively by using developed chromatographic conditions. Fig 2 to 4 represent the chromatograms of standard, negative swab control and resolution solution.

Fig. 2: Negative swab control chromatogram

Fig. 3: Standard chromatogram

Fig. 4: Resolution solution chromatogram

3.3 Optimization of sample treatment

Cotton swabs were spiked with different quantities of ketorolac tromethamine and Placed in glass tubes. After addition of different solvents and their mixtures (water, methanol and mobile phase), the tubes were sonicated for different times (5, 10, 15 and 30 minutes) and the solutions were analyzed by HPLC. The optimum conditions were achieved with mixture of water and methanol (55: 45, v/v) and a sonication time of 10 minutes. In all the cases, the best results were obtained using two cotton swabs (the first wetted with diluent and the second dry). Hence this technique was applied in the subsequent work.

Some of the results obtained for swab samples from different locations within the manufacturing equipement are summarized in table I.

Table I: Results obtained from analysis of Ketorolac tromethamine in swab samples collected from different locations within the equipment

Equipment swabbed	Location swabbed	Ketorolac tromethamine detected (µg per swab)
Material dispensing scoops	Internal surface	< LOQ
Material dispensing scoops	External surface	< LOQ
Weighing balance	Weighing pan	< LOQ
Manufacturing vessel	Top surface	< LOQ
	Middle surface	< LOQ
	Bottom surface	0.20 ( <LSA)
Transfer tube	Line starting point	< LOQ
Transfer tube	Line End point	< LOQ

3.4 Validation of analytical method

Once the chromatographic conditions had been optimized, the method was validated for linearity, precision, accuracy, LOD and LOQ, selectivity, solution stability.

3.4.1 System suitability test:

System suitability test is essential for the assurance of the quality performance of a chromatographic system. Standard of 10 µg/ml was injected five times into HPLC as per test method. RSD of area of Ketorolac peak from five injections was found 0.53%. Theoretical plates of Ketorolac peak was found to be 12115. USP tailing of Ketorolac peak was found to be 1.09. The results are summarized in table II.

3.4.2 Selectivity:

The selectivity of the method was checked by injecting Ketorolac tromethamine standard solution, Resolution solution, background control sample, Positive swab control sample and negative swab control sample into HPLC with PDA detector as per test method. There was no interference at retention time of Ketorolac due to back ground control sample and the negative swab control samples. Ketorolac and its impurities (1-hydroxy analogue and 1-keto analogue) peaks were found well separated. Peak purity of Ketorolac peak was passed in standard, Resolution solution and positive swab control sample.

3.4.3 LOD and LOQ:

LOD and LOQ were determined based on the standard deviation of the response and the slope of the calibration curve at low concentration levels according to ICH guidelines. The LOD and LOQ for Ketorolac were found to be 0.050 µg/ml and 0.130 µg/ml respectively. The results are summarized in table II.

3.4.4 Linearity:

Linearity of the test method was conducted by injecting Ketorolac tromethamine solution in the concentration range of 0.130–30 µg/ml. Plotted a linearity graph of concentration versus area for Ketorolac. The correlation co-efficient, slope, intercept and bias at 100% response are summarized in table II and See fig 5.

3.4.5 Method precision:

The precision of the test method was performed by spiking Ketorolac tromethamine solution on to stainless steel plate to achieve final solution containing 10 µg/ml. This study was assessed by comparing the amount of analyte determined versus the known amount spiked for six replicates. The RSD of % recovery of six sample preparations was found 2.20%. The results are summarized in table II.

3.4.6 Accuracy:

The recovery was assessed by comparing the amount of analyte determined versus the known amount spiked at three different concentration levels (5.0 µg/ml, 10 µg/ml and 15 µg/ml) with 3 replicates (n=3). The accuracy was determined by spiking ketorolac tromethamine on stainless steel plates, performed swabbing and analyzed as per test method. The recovery at three different concentrations was found more than 85%. The results are summarized in table III.

Table II: Results of System suitability, LOD /LOQ, Linearity and Method precison

Parameters

Ketorolac tromethamine

System suitability

a) USP tailing factor ( < 2.0)

b) USP plate count (> 3000)

c) % RSD of five standard (< 2.0%)

1.09

12115

0.53

LOD and LOQ values

a) LOD value

b) LOQ value

0.052 µg/ml

0.130 µg/ml

Linearity

a) Correlation coefficient (r)

(NLT 0.999)

b) Linearity Equation

c) Bias at 100 % response

(NMT 2.0)

0.99995

Y = 50356.X + 356.5

0.07

Method Precision (Repeatability)

a) Mean % Recovery (n = 6)

( > 80.0%)

b) % RSD (n = 6) (NMT 5.0%)

97.60 %

2.20

Table III: Results of Accuracy

Amount added µg/ml	Amount found µg/ml	Recovery (%) ( > 80%)	% RSD (n = 3)
5.22	5.11	97.9	1.12
10.45	10.04	96.1	1.79
15.67	14.19	90.6	5.33

Table IV: Results of solution stability

Time

% Assay

% Assay diff.(< 3.0%)

USP Tailing(< 2.0)

USP plate count(>3000)

Standard

Initial

Day 1

Day 2

100.0

98.7

100.0

0.0

1.3

0.0

< 2.0

> 3000

Test sample

Initial

Day 1

Day 2

98.5

100.1

99.8

0.0

1.6

1.3

< 2.0

> 3000

Fig 5: Linearity plot of Ketorolac tromethamine

3.4.7 Solution stability of standard and sample solution:

Prepared standard solution and positive swab control sample as per test method and kept on bench top at room tempearature. Injected standard and sample solutions into HPLC as per test method at initial, after 1 day and after 2 days. Calculated % assay of standard and test solution against fresh standard. The assay of standard and sample was found satisfactory from initial to 2 days. This indicate that standard and sample preparation (Positive swab samples) are stable for 2 days at room temperature. The results are summarized in table IV.

4. CONCLUSION:

A simple, sensitive and accurate isocratic HPLC method has been developed for determination of Ketorolac tromethamine residues on the surface of manufacturing equipment. Validation of the method revealed that method is selective, precise, linear and accurate. Extraction of Ketorolac tromethamine residues from surface by using two cotton swabs, the first wet with methanol and the second dry, is recommended. The recovery of Ketorolac tromethamine obtained from swabs was found > 85% for three different concentrations. The solution stability data revealed that standard and swab samples are stable for 2 days at room temperature. The overall procedure can be used to determine trace levels of Ketrolac tromethamine residues in production equipment area to confirm the efficiency of the cleaning procedure in pharmaceutical industries.

5. AKNOWLEDGEMENT:

The authors are very thankful to Dr. Reddy’s Laboratories, Hyderabad, India for providing research facilities for this work. They are also very thankful to Dr. Madhusudan of R &D, Dermatology, Dr. Reddy’s laboratories, India for providing samples and materials for this research work.

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