Quantitative Determination of Penicillin Traces in Non-Penicillin Drug

Journal of Chromatographic Science, Volume XX, Number XX July 2015

QuantitativeDetermination of Penicillin Traces in Non-Penicillin Drug Productusing UPLC

QuantitativeDetermination of Penicillin Traces in Non-Penicillin Drug Productusing UPLC

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Abstract

A novel quantitative analytical method for determination of penicillin contaminant (Amoxicillin) in non-penicillin pharmaceutical drug product (Ibuprofen tablets 400 mg) has been developed and validated using Ultra Performance Liquid Chromatography (UPLC). Extraction of Amoxicillin from the drug product was carried out in dist. Water and the separation was achieved using BEH C18 column with a particle size of 1.7 µm (100 mm × 2.1 mm). The Isocratic run accomplished using Phosphate Buffer (pH=5.0): Methanol (95:5, v/v) as mobile phase at a flow rate of 0.3 ml/min. The rapid, accurate and low cost UPLC method was proven to be suitable within the current good manufacturing practices (cGMP) of the pharmaceutical. Validation was conducted on the developed method as per the ICH guidelines Q2 (R1) and Q2B (e-f). The method was linear in the range of (0.024 – 0.0096 µg/mL for amoxicillin) with correlation coefficient r2 = 0.999 and net in terms of specificity, linearity, precision, accuracy, quantitation limit and detection limit, and appeared satisfactory. The precision assessed in terms of injections repeatability with max. % RSD=1.8%, Intermediate precision Day-1 with %RSD= 0.96, intermediate precision Day-2 with %RSD=1.56.The method was successfully applied to pharmaceutical quality control laboratory and fulfilled the regulatory requirements.

Key words: UPLC, Quantitative Determination, Penicillin Traces, non-penicillin drug, Design of Experiment.

Penicillin is one of the most well-known beta-lactam antibiotic drugs used for treatment of bacterial infections in humans and animals (1-3). Controversy, it can be a sensitizing agent that triggers a hypersensitive exaggerated allergic immune response in some people. Common&nbspallergic reactions&nbspto penicillin include&nbsprashes,&nbsphives, itchy&nbspeyes, swollen lips,&nbsptongue and face. The most serious allergic reaction to penicillin is an anaphylactic reaction which can be life threatening (4-7).&nbspAccordingly, implementing regulatory requirements for preventing cross-contamination with penicillin is a key element of manufacturing sites.&nbsp

In the pharmaceutical industry, one of the general concerns lies in the cross-contamination of penicillin into non-penicillin products especially when both are manufactured in the same building. FDA (Food and Drug Administration) strictly regulated the manufacturer in different parts of the 21 CFR 211 (current Good Manufacturing Practice for Finished Pharmaceuticals). It is recommended to all penicillin manufacturers to establish a stringent control including separate facilities, air handling system and dedicated equipment for penicillin products. Furthermore, if a reasonable possibility exists that a non-penicillin drug product has been exposed to cross-contamination with penicillin, the non-penicillin product shall be tested for the presence of penicillin and such drug product shall not be marketed if detectable levels are found (8). In view of the above mentioned facts, it is essential to establish an analytical method for detection of penicillin traces on the occasion of manufacturing of non-penicillin products in the facility exposed to penicillin. The pressing need that the method shall have a detection limit not to exceed the safe allowable tolerance level set by the FDA (10ppb for Amoxicillin). (9)

Different analytical methods were reported for determination of penicillin traces in milk (10-17), in food (18-25) and animal tissues (26-28) and in groundwater (29) to avoid the adverse impact of penicillin contamination. On the other hand, the reported methods for detection of penicillin residues in the pharmaceutical field mainly focus on the cleaning validation (30).

However, only one papers can be found for determination of penicillin in non-penicillin drug products using HPLC coupled with Tandem Mass Spectrometry (31) and no paper can be found in the literatures in which UPLC was used for the determination of penicillin traces (Amoxicillin) in non-penicillin drug products in pharmaceutical industry.

Experimental

Chemicals and Reagents

The API Amoxicillin Trihydrate (AM-SN-09966) was supplied by ABC pharmaceutical Co., Malaysia. O-Phosphoric Acid (Code: O/0500/PB17) and Potassium Dihydrogen Phosphate (Code: P/4800/53), Analytical reagent grade, were obtained from Fischer Scientific, UK. Methanol (Product No. 20864.320), HPLC Grade, was obtained from BDH, UK. Buffer Solutions with accuracy +/- 0.02 @ 20°C (pH = 4.00, code: 272168) and (pH = 7.00 code: 272170) were obtained from Pancreas, Spain

Equipment

Acquity Ultra Performance Liquid Chromatographic (UPLC) System (Waters, Milford, MA, USA) was used to run the analysis. The system was equipped with Binary Solvent Manager as a pump, Sample Manager as auto-injector, Tunable TUV Detector used for UV detection and Empower 3 for control and data handling.

Seven Multi pH meter (Mettler Toledo, USA ) was used for pH adjustment, Ultrasonic Bath-MXB6 (Grant, UK ) was utilized for sonication of standard and sample , Analytical balance- XP26 (Mettler Toledo, USA ) was used in weighing , Centrifuge-Z-300 (Hermle, Germany ), Mechanical Shaker –SM 30 (Edmund Buhler, Germany )and Vortex Mixer-Whirli (Grant, UK) were used during sample preparations. Minitab 17 Statistical Software was used for data analysis.

Chromatographic Condition

The UPLC column was used of Waters Acquity BEH C18 (2.1 x100mmx1 .7µm particle size), the temperature of the column maintained at 35° C. The flow mode of the mobile phase was isocratic at flow rate of 0.3 ml/min. and composition of 95:5 (v/v) of Phosphate buffer (pH=5.0): Methanol. The mobile phase filtered using 0.22- µm Millipore Nylon membrane filter and degassed for 20 min. using ultrasonic bath. The injection volume was 5 µl using partial needle overfill as injection mode. The weak needle Wash limited to 10% methanol while the strong needle wash extended to 70% methanol. The peaks detection was monitored at wavelength 230nm and setting the filter time constant at 1.25 sec.

Standard Preparation

Weigh accurately on an analytical balance about 60 mg Amoxicillin Trihydrate RS into 100 ml volumetric flask, add 50 ml bi-distilled water and shake for 5 minutes using the mechanical shaker, sonicate for 5 minutes using ultrasonic water bath then Dilute to volume using bi-distilled water and mix well ( Stock solution of 0.60 mg/ml).

Further dilutions were prepared from the stock solution to have 0.024, 0.036, 0.048, 0.060, 0.072, 0.084 and 0.096 µg/ml of Amoxicillin in bi-distilled water (representing 40 – 160 %). Filter the solutions into 2ml vial using Acrodisc GHP 0.2 µm syringe filter.

Sample Preparation

Insert 10 tablets of Ibuprofen 400 mg in 25ml centrifuging tube. Spike with 20 ml of Amoxicillin trihydrate RS at concentration levels of (100%) of standard solution and shake using the vortex mixer for 5 minutes then centrifuge at 2000 rpm. Filter into 2ml vial using Acrodisc GHP 0.2 µm syringe filter (Spiked Sample Solution for precision).

In each of 9 centrifuging tube (25ml), insert 10 tablets of Ibuprofen 400 mg. Spike individually 3 x 3 replicate of Amoxicillin Trihydrate (20ml) at concentration levels of (80%), (100%), and (120%) of working Standard Solution, shake using the vortex mixer for 5 minutes then centrifuge at 2000 rpm . Filter into 2ml vial using Acrodisc GHP 0.2 µm syringe filter, (Spiked Sample Solution for Accuracy/Recovery).

Development of the Method

The target of the method set to develop the UPLC method using isocratic elution mode for detection of the drug traces &quotAmoxicillin&quot in the presence of the matrix&quot Ibuprofen tablets 400mg&quot with acceptable resolution and detectable level. The mobile phase that often used in the separation of the drug in the official compendial method &quotUSP/NF (32) or EP” (33) is the mixture of phosphate buffer &quotpH=5.0” and acetonitrile in different composition ratio. Using acetonitrile leads to noticeable level of interference but enough separation obtained upon replacing with methanol. The drug detection was best found at 230nm wavelength. In addition, the calibration graphs achieved at 230 nm show good correlation coefficient. Different pH values of the buffer used in the mobile phase preparation gives good recovery but the run time was getting longer. To solve this problem, several mobile phases were tested, which was accomplished by varying their pH and composition. This was done to obtain the desired chromatographic separation. The proposed mobile phase composed by composition of 95:5 (v/v) of Phosphate buffer (pH=5.0): Methanol where the buffer prepared by weighing 2.72 g Potassium Dihydrogen Phosphate in 1L bi-distilled water, adjust pH to 5.0 using ortho-Phosphoric Acid An UPLC™ BEH C18 (100 × 2.1 mm2) 1.7 μm column was selected to achieve good peak shape and symmetry. However, shorter column of 50mm in length did not provide the optimum separation. The injection carried out in the UPLC with different volume ranging from 2 to 10 µl and the best choice was set to 5 µl as the larger the volume the broader the peak shape with no positive impact on the s/n ratio. The flow rate of the mobile phase was kept 0.3 mL / min and the column temperature was maintained at 35°C. DOE for the robustness of the method using Minitab 17 software was implemented and effect of different variables was studied to reach to the optimum selected conditions.

Method Validation

Validation of the developed analytical method was conducted as per the ICH guidelines Q2 (R1) (34). The validation characteristics considered for evaluation are specificity, Linearity, range, accuracy, LOQ, LOD, precision, and robustness.

Specificity

Specificity was carried out to evaluate the matrix effect of &quotIbuprofen tablets 400 mg&quot on the analyte &quotAmoxicillin&quot. The test was established by comparison of the chromatograms of the standard solution of Amoxicillin with concentration of 0.060 µg/ml (100 %) in bi-distilled water and the sample solution prepared by dissolving 10 unites of the Ibuprofen tablets into 20 ml bi-distilled water. shake using the vortex mixer for 5 minutes then centrifuge at 2000 rpm . Filter into 2ml vial using Acrodisc GHP 0.2 µm syringe filter. The discrimination of the analyte was confirmed with no interference.

Linearity and Range

A linearity study verifies that the sample solutions are in a concentration range where analyte response is linearly proportional to concentration.

The linearity and range of the method were determined at seven concentration levels ranging from 40- 160 % (0.024 – 0.096 µg/mL for amoxicillin). The calibration curves were constructed by plotting peak areas versus concentration of Amoxicillin. The slope, Y-intercept and correlation coefficient were calculated.

Accuracy

Accuracy is the measure of how close the experimental value to the true value. It was assessed by a recovery percentage of Amoxicillin 100 % (0.060 µg/ml) in 9 determinations at the level of 80%, 100%, and 120% of the target concentration (3 preparations for each of 0.048 µg/ml, 0.06 µg/ml, and 0.084 µg/ml) using 10 unites of Ibuprofen 400 mg tablets spiked with the corresponding amounts of analyte. At each recommended level studied, replicate samples are evaluated. The mean of the replicates, which is expressed as % label claim, shows accuracy of the method used to perform the test.

Precision

Precision of the method was evaluated using the repeatability and intermediate precision. Repeatability/System precision was assessed by calculating the relative standard deviation (% RSD) for 5 determinations (n=5) using the 7 concentration levels of Amoxicillin standard (40- 160 % equivalent to 0.024 – 0.096 µg/mL). On the other hand, Intermediate precision was carried out to check the reliability of the method in a different environment other than that used during development of the method. It was determined by computing the RSD for six injected sample solutions (n=6) that was prepared from one batch of Ibuprofen tablets 400 mg tablets spiked with 100 % conc. Level of Amoxicillin. The same procedure was implemented in different day to evaluate the ruggedness of the method.

System Suitability Test

The system suitability test is parameters that provide assistance in achieving the purpose of accuracy and precision of data collected from well-behaved chromatographic system. The five parameters considered in this test were the capacity factor, precision/injection repeatability, tailing factor, theoretical plates and resolution. The test carried out by injecting the standard solution of the Amoxicillin (100% or 0.06 µg/ml) and the fortified samples with the same conc. of the standard.

Detection Limit and Quantitation Limit

The Detection limit (DL) and Quantitation Limit (QL) of the method were calculated by using the standard deviation of the response (σ) and the slope of the calibration curve (S) as derived from the linearity plot. The DL and QL were estimated from three calibration curve using the following formula

(i). Detection Limit = 3.3 σ

S

(ii). Quantitation Limit = 10 σ

S

Robustness

It shows the reliability of the analysis with respect to deliberate variations in method parameters. DOE (Design of Experiment) was used to assess the robustness of the method. Four factors were considered in evaluation of the robustness of the method such as influences of variation in pH of the mobile phase, changes in composition of the mobile phase variation in the temperature of column and variation in flow rate.

Run the protocol of the experiment to get the response for the %Assay) for the samples as prepared in method of the precision

Results and Discussion

Specificity

The chromatograms displayed in Figure 1 demonstrate a retention time of 1.955 min. for the analyte peak &quotAmoxicillin&quot with no interference from the matrix &quotIbuprofen Tablets 400 mg&quot.

Figure 1. Specificity of the method

Linearity and Range

The response of the drug was found to be linear in the investigation concentration range and the linear regression equation for Amoxicillin was Y = 19768X + 11.21 with correlation coefficient R2= 0.9995 (Figure 2). Where X is the concentration in µg/ml and Y is the peak area response. Statistical analysis of the data as shown in table I confirmed the linearity of method.

Figure 2. Linearity of Amoxicillin

Detection Limit and Quantitation Limit

Table II is showing the regression data of the three calibration curves. The detection limit and quantitation limit were determined to be 0.008 µg / ml and 0.025 µg / ml respectively. Accordingly the results fulfill the requirements of the FDA to be detectable in a level less than the safe level of Amoxicillin &quot10 ppb&quot.

Accuracy

The mean % recovery found to be 101.7, 99.7 and 97.9 respectively while the %RSD at each level found to be 4.6, 0.5 and 1.2 respectively. The obtained individual test results for the accuracy are well within the set criteria as indicated in Table III.

Precision

Table IV shows the observed % RSD for the peak area of the repeatability test. The obtained results ranges from RSD= 0.7 to 1.8 % which is well within the specified limit. Figure 3 is displaying the repeatability chromatograms for the analyte at the conc. Level of 100%.The RSD for intermediate precision was 0.96 % for the day-1 of the analysis, 1.56 for Day-2 and 1.97 between the two days. Table IV shows the obtained test results.

Figure 3. Repeatability of the Method at 100% conc. Level of Amoxicillin

System Suitability Test

The displayed test results convey satisfaction of system suitability test to the set criteria. Figure 4 &amp 5 are showing the obtained chromatograms and test results of each parameter.

Figure 4. System Suitability Test

Figure 5. Chromatogram of Resolution for System Suitability Test

Robustness

The DOE using Minitab 17 Software was used to study the main effect of each factor and the interactions of the factors to eliminate the factors that do not have impact on y response.

The steps for evaluation start with the identification of the factors. Four factors are considered as indicated in Table V. The two level designs were used for each factor as shown Table VI.

The 1/2 fractional factorial design shown in Figure 6 was selected in which the total number of runs reduced from 16 to 8 with random order and resolution IV (Figure 7) as stated in Table VII

The results of the protocol of the experiment are shown in Table VIII.

Figure 6. Fractional Factorial Design

Figure 7. Available Factorial Design (with resolution).

By analyzing the obtained data to verify to effect of factor (main and interaction effect). It was clear from the Pareto chart and normal plot of the effect (Figure 8) that there is no significant factor that may affect the run under the set variables of conditions.

Figure 8. Pareto Chart and Normal Plot for factors effect.

Conclusion

A quantitative determination method for trace amount of penicillin contaminants in a commercially available drug product has been established. Detection was achievable at the level of 0.008 µg/ml for Amoxicillin in Ibuprofen tablets 400 mg. Validation of the established method was made and shown to be reasonable. This fulfilled the regulatory requirements by the authorities. It can be concluded that the developed method has higher selectivity and sensitivity than other reported methods in the pharmaceutical industries that are running the Amoxicillin and Ibuprofen tablets 400mg in separate area of the same plant.

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Tables

Table I: Regression analysis

Standard Name

Lot #

Potency ( % )

Wt (mg)

Amoxicillin Trihydrate

AM2013061222

99.50

60.22

&nbsp

Conc. (%)

Conc. (µg/ml)

Peak Area

Trial I

Trial II

Trial III

Average

40%

0.024

510

500

492

501

60%

0.036

721

694

707

707

80%

0.048

944

939

949

944

100%

0.060

1230

1196

1191

1206

120%

0.072

1463

1443

1458

1455

140%

0.084

1663

1654

1651

1656

160%

0.096

1918

1906

1913

1912

REGRESSION DATA

x-Coefficient [b]

19768

Constant [a]

11.2143

Correlation Coefficient

0.999525

Table II: Detection and quantitation limit

x-Coefficient [b]

Trial I

19723.2

Trial II

19767.9

Trial III

19821.4

Average (S)

19770.8

SD (σ)

49.17

% RSD

0.25%

Table III: Accuracy

Accuracy ( % Recovery)

&nbsp

&nbsp

&nbsp

Standard Information

Standard Name

Lot #

Potency ( % )

Wt (mg)

Conc. (µg/ml)

Peak Area

Amoxicillin Trihydrate

AM2013061222

99.50

58.23

0.0579

1151

&nbsp

1104

1120

&nbsp

Average

1125

SD (σ)

23.90

% RSD 2.1

Sample Information

Trial #

Amount Spikked (%)

Peak Area

Standard Conc. (µg/ml)

Conc. Found in Samples (µg/ml)

% Recovery

Average

1

80%

990

0.0480

0.0510

106.3

101.7

950

0.0480

0.0489

101.9

902

0.0480

0.0465

96.9

SD

4.70

% RSD

4.6

2

100%

1127

0.0584

0.0580

99.3

99.7

1130

0.0584

0.0582

99.7

1136

0.0584

0.0585

100.2

SD

0.45

% RSD

0.5

3

120%

1357

0.0720

0.0699

97.1

97.6

1353

0.0720

0.0697

96.8

1382

0.0720

0.0712

98.9

SD

1.14

% RSD

1.2

Average

99.6667

SD

2.0502

% CV

2.06 %

Table IV: Precision

Standard Name

Lot #

Potency ( % )

Wt (mg)

Amoxicillin Trihydrate

AM2013061222

99.50

60.22

Repeatability

Conc. Level (µg/ml)

0.024

0.036

0.048

0.060

0.072

0.084

0.096

Peak Area

510

721

944

1230

1463

1663

1918

500

694

939

1196

1443

1654

1906

492

707

949

1191

1458

1651

1913

507

727

956

1173

1463

1626

1923

496

710

964

1204

1446

1708

1870

&nbsp

&nbsp

&nbsp

&nbsp

&nbsp

&nbsp

&nbsp

Average

501

712

950

1199

1455

1660

1906

SD

7.48

12.83

9.86

20.83

9.50

29.94

21.08

%RSD

1.5

1.8

1.0

1.7

0.7

1.8

1.1

Table V: Identification of the factors

&nbsp

Factors

Factors ID

Low

High

Unit

Type

1

pH of the Mobile Phase

A

4.5

5.5

&nbsp

QN

2

Composition of the Mobile Phase

B

97:3

93:7

v/v

QN

3

Temperature of the Column

C

30

40

°C

QN

4

Flow Rate

D

0.2

0.4

ml

QN

Table VI: Definition of levels of each factor

Factors ID

Low

High

A

-1

1

B

-1

1

C

-1

1

D

-1

1

Table VII: I / 2 fractional factorial design

StdOrder

RunOrder

CenterPt

Blocks

A

B

C

D

3

1

1

1

-1

1

-1

1

8

2

1

1

1

1

1

1

7

3

1

1

-1

1

1

-1

2

4

1

1

1

-1

-1

1

6

5

1

1

1

-1

1

-1

1

6

1

1

-1

-1

-1

-1

4

7

1

1

1

1

-1

-1

5

8

1

1

-1

-1

1

1

Table VIII: Protocol and response

Std Order

pH of the Mobile Phase

Composition of the Mobile Phase

Temperature of the Column

Flow Rate

% Assay

3

4.5

93:7

30

0.4

102.6

8

5.5

93:7

40

0.4

103.6

7

4.5

93:7

40

0.2

101.1

2

5.5

97:3

40

0.4

101.9

6

4.5

97:3

30

0.2

98.3

1

5.5

97:3

40

0.2

99.7

4

4.5

93:7

30

0.2

100.2

5

4.5

97:3

40

0.4

103.8

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