System Suitability For Cefuroxime

Published Date: 02 Nov 2017

Chapter 5

System suitability was carried out for determination of instrument performance for the following parameters which include sensitivity, chromatographic retention by analysis of Cefuroxime sodium and Cefuroxime d3 as an internal standard prior to application of method.The following result was obtained during analysis. The percent coefficient of variation for peak area ratio of analyte to internal standard was found to be 2.183 & 2.335 respectively.It was concluded that the peak area ratio of analyte to IS were within linearity of the acceptance criteria on the basis of results.

Table 5.1: System Suitability for Cefuroxime

SYSTEM SUITABILITY TEST RECORD

PROJECT / STUDY NO.:

BL-BMV-035

DATE:

15\JUN\12

ANALYTE:

Cefuroxime

COLUMN ID:

BLC-247

INJ. NO

SAMPLE ID

RETENTION TIME

ANALYTE AREA

IS AREA

AREA RATIO

ANALYTE

IS

1

SS-01-01

1.09

1.09

486944

179808

2.708

2

SS-01-02

1.09

1.09

480871

179393

2.681

3

SS-01-03

1.09

1.09

482648

179787

2.685

4

SS-01-04

1.09

1.09

488322

184642

2.645

5

SS-01-05

1.09

1.09

485856

181686

2.674

6

SS-01-06

1.09

1.09

475417

179068

2.655

Mean

2.6747

SD

0.02247

%CV

0.84

Figure 5.1: Mass Spectra for System Suitability of Cefuroxime (Analyte)

Acceptance Criteria:

The % C.V. for peak area ratio of analyte or metabolite to internal standard should be less than 3% in LC-MS/MS.

Method Validation Results:

Selectivity (Specificity) :

The selectivity is generally defined as the lack of interfering peaks at the retention time of the assayed drugs and the internal standard in the chromatograms. Blank sample of the appropriate biological matrix obtained from six different sources (subject or donor). For analysis purpose, processed and inject all the six blank plasma lots and then inject LLOQ samples spiked in respective blank plasma lots. Inject reference solution (a mixture of drug and internal standard in appropriate concentration to check the respective retention time, chromatography and area response).

Six different plasma lots were analysed for selectivity.

Sequence of Injection: set of six injections including, Blank and LLOQ

Selectivity is calculated as per the formula given below:

Blank Area

% Interference = × 100

Analyte or IS Area

Table 5.3: Selectivity for Cefuroxime

Sr. No

Matrix Lot No.

Analyte Area

IS Area

Blank

LLOQ

% Interference

Blank

IS Area

% Interference

1

XXX-01

0

32078

0

0

88018

0

2

XXX-02

0

29764

0

0

74671

0

3

XXX-03

0

29539

0

0

78317

0

4

XXX-04

0

29837

0

0

79122

0

5

XXX-05

0

27634

0

0

75804

0

6

XXX-06

0

31841

0

0

81819

0

7

XXX-07

(Haemolysed)

0

31218

0

0

81887

0

8

XXX-08

(Lipemic)

0

30704

0

0

76666

0

Acceptance Criteria:

Response of interfering peaks at the retention time of analyte must be ≤ 20% of the individual response of the processed LLOQ.

Response of interfering peaks at the retention time of internal standard is ≤ 5% of the individual response of the processed LLOQ.

Blank

LLOQ

Figure 5.4: Mass Spectra for Selectivity of Cefuroxime (Analyte)

Sensitivity

Sensitivity is measured in terms of LLOQ-QC (lower limit of quantitation). Prepared six plasma spiked LLOQ-QC samples using the same spiking dilution used to prepare LLOQ-QC of quality control sample. These six LLOQ-QC samples processed and inject along with calibration curve standards in same range used for calculation of precision and accuracy. Sensitivity was measured by calculating the concentration of the LLOQ-QC sample against calibration curve standard.

Table 5.5: Sensitivity for Cefuroxime

LLOQ-QC

Nominal Concentration (ng/ml)

3.112

Acceptance Limit (ng/ml)

2.490

3.734

LLOQ

Calculated Concentration

1

3.168

2

3.549

3

3.126

4

3.297

5

3.452

6

3.364

Mean

3.326

S.D. (+/-)

0.163

% C.V.

4.901

% Nominal

106.877

LLOQ- Cefuroxime

Figure 5.7: Mass Spectra for Sensitivity of Cefuroxime (Analyte)

Acceptance Criteria:

The accuracy of the mean concentration of LLOQ-QC sample must be within ±20% of the nominal concentration.

The % CV should be ≤ 20%.

At least 67% of the LLOQ-QC samples should meet the acceptance criteria for accuracy.

Linearity

Evaluate at least 3 calibration curves generated using spiked samples to establish linearity. The matrix based standard curve should consist of minimum of 8 standard points excluding blanks.Select the simplest regression algorithm with minimum weighting factor. Use all calibration curves for the determination of mean concentrations, S.D., CV and % nominal all points of calibration curves, using the chosen regression algorithm.Do not include standard blank and standard zero in the calibration curve.Do not remove the accepted standard from calibration curve.Tabulate the results of calibration curve parameters e.g. slope, intercept and coefficient of correlation.

Table 5.7: Linearity of Calibration Curve (CC) Standards for Cefuroxime

CC Point

Analyte conc. (ng/ml)

Replicates conc. (ng/ml)

Mean

S.D.

%C.V

%Nominal

1

2

3

CC 1

3.07

3.15

3.24

3.18

3.19

0.045

1.434

103.93

CC-2

6.14

6.08

5.37

6.02

5.82

0.395

6.788

94.78

CC-3

30.72

32.45

33.00

32.58

32.68

0.289

0.885

106.36

CC-4

61.45

63.57

60.27

65.98

63.27

2.863

4.525

102.96

CC-5

87.79

90.38

89.69

93.15

91.07

1.833

2.013

103.74

CC-6

125.4

128.67

129.54

127.72

128.64

0.908

0.706

102.57

CC-7

179.17

187.90

188.83

190.25

188.99

1.181

0.625

105.48

CC-8

199.07

198.97

196.35

198.71

198.01

1.444

0.729

99.46

Table 5.8: Concentration Response Linearity of Calibration Curve Standards for Cefuroxime

Sr. No.

Sample

Analyte Conc. (ng/ml)

Analyte Area

1

CC-1

3.07

1908

2

CC-2

6.14

3850

3

CC-3

30.73

21730

4

CC-4

61.45

46780

5

CC-5

87.79

52364

6

CC-6

125.42

82819

7

CC-7

179.17

107455

8

CC-8

199.08

120095

Figure 5.8: Concentration Response Linearity of Calibration Curve Standards for Cefuroxime

CC-1 CC-2

CC-3 CC-4

CC-5 CC-6

CC-7 CC-8

Figure 5.9: Mass Spectra for Linearity of Cefuroxime (Analyte)

CC Point

Analyte conc. (ng/ml)

Replicates conc. (ng/ml)

Mean

S.D.

% C.V

% Nominal

1

2

3

LLOQ

3.11

3.08

3.27

2.96

3.10

0.15

4.96

99.71

LQC

8.89

8.77

8.64

8.86

8.76

0.11

1.29

98.49

MQC

59.28

59.04

59.56

59.33

59.31

0.26

0.44

100.04

HQC

169.39

169.99

169.36

169.29

169.21

0.20

0.12

99.90

Table 5.11: Linearity of Quality Control (QC) Standards for cefuroxime

Sr. No.

Sample

Analyte Conc. (ng/ml)

Analyte Area

1

LLOQ

3.11

1626

2

LQC

8.89

5266

3

MQC

59.28

33211

4

HQC

169.39

91889

Table 5.12: Concentration Response Linearity of Quality Control Standards for Cefuroxime

Figure 5.12: Concentration Response Linearity of Quality Control Standards for Cefuroxime

LQC- Cefuroxime

MQC- Cefuroxime

HQC- Cefuroxime

Figure 5.14: Mass Spectra for Quality Control Samples

Acceptance Criteria:

For Standard Blank and Standard Zero, the percentage interference at the retention time of the analyte(s) should not be more than 20% of the accepted LLOQ standard response and for Standard Blank; percentage interference at the retention time of the internal standard should not be more than 5% of the mean of the accepted CC standards ISTD response

For both Standard Blank and Standard Zero, at least one of the two runs should meet the defined acceptance criteria.

Accept calibration curve standard when, the back calculated values for CC standards are defined acceptance criteria within ± 15% of the nominal concentration, except LLOQ where the back calculated value shall be within ± 20% of the nominal concentration.

Consider only accepted standards for construction of calibration curve.

Accept the CC only if 75% of the CC standards are acceptable i.e. a minimum of 6 out of 8 non-zero standards (including LLOQ and ULOQ).

Accept calibration curve standard when, the back calculated concentration value for CC standard are within ±15% of the nominal concentration, except for LLOQ, where the back calculated concentration value shall be within ± 20% of the nominal concentration.

Accuracy & Precision

The precision and accuracy (P&A) data were obtained by analyzing three different precision and accuracy batches which consist of following set of sample:

System suitability sample

Blank

Blank + Internal Standard

Spiked calibration standard of atleast 6 non-zero conc. (CC-1 to CC-8)

LLOQ-QC (6 Samples)

Lower –QC (6 Samples)

Middle-QC (6 Samples)

Higher-QC (6 Samples)

Table 5.15: Precision & Accuracy for Cefuroxime (Intra-day)

Quality Control Samples

LLOQ

LQC

MQC

HQC

Analyte concentration

3.112

8.893

59.285

169.385

Batch-1

1

2.578

7.165

55.343

169.631

2

2.808

7.797

56.635

158.057

3

3.396

7.202

56.147

169.884

4

2.918

7.620

21.381

152.743

5

3.210

7.852

54.733

163.163

6

2.905

7.149

56.406

169.304

Batch-2

7

3.011

8.964

58.996

169.112

8

3.164

8.156

59.116

169.354

9

3.052

8.583

59.215

169.325

10

2.968

8.997

59.322

169.286

11

3.112

8.264

59.155

169.052

12

3.138

8.448

59.281

169.234

Batch-3

13

2.859

8.981

59.311

169.594

14

2.967

8.902

59.059

169.236

15

3.029

8.631

59.187

169.382

16

2.891

8.785

59.293

169.358

17

3.123

8.959

59.087

169.399

18

3.067

8.521

59.007

169.289

Mean

3.011

8.276

57.815

167.46

S.D.

0.179

0.661

2.240

4.718

% C.V.

5.947

7.988

3.875

2.817

% Nominal

96.75

93.06

97.52

96.86

Table 5.17: Precision & Accuracy for Cefuroxime (Inter day)

Quality Control Samples

LLOQ

LQC

MQC

HQC

Analyte concentration

3.112

8.893

59.285

169.385

Batch-1

1

2.987

8.568

58.964

169.784

2

3.999

8.631

59.311

169.022

3

3.128

8.994

59.112

168.845

4

3.225

8.842

59.436

168.247

5

2.986

8.654

59.258

163.258

6

2.831

8.723

59.069

169.347

Batch-2

7

3.356

8.954

59.321

169.994

8

3.116

8.263

58.846

169.783

9

3.058

9.021

59.222

169.542

10

3.254

8.578

59.671

169.648

11

3.031

8.967

59.349

168.726

12

3.139

8.911

58.129

168.129

Batch-3

13

2.978

8.973

58.843

170.067

14

3.079

8.856

59.054

169.883

15

3.167

8.598

58.942

169.951

16

3.264

8.712

59.146

167.328

17

3.041

8.887

59.785

169.654

18

2.959

8.934

58.741

169.753

Mean

3.139

8.781

59.067

168.942

S.D.

0.245

0.203

0.396

1.609

% C.V.

7.812

2.311

0.670

0.952

% Nominal

100.86

98.74

99.63

99.73

Acceptance Criteria:

Precision: For between and within batch CVs for low, medium and high concentration must be ≤ 15% and for LLOQ-QC, it must be ≤ 20%, using minimum three batches.

Accuracy: For between and within batch mean value must be within ± 15 % of nominal value at low, medium and high QC concentration and at LLOQ-QC it must be within ± 20%.

At least 67% (four out of six) of total quality control sample and at least 50% quality control sample at each level should meet above acceptance criteria.

Matrix Effect

Matrix effect studied by comparing the response of post extracted samples spiked after extraction with the response of the aqueous sample. Matrix effect evaluated for six lots of plasma which were processed and then spiked post extraction after elution in duplicate and finally reconstituted with aqueous dilutions of LQC, MQC, and HQC along with internal standard. The following result was obtained during analysis. By looking after the result of analysis, it was concluded that the matrix effect ratio for both post-extracted and aqueous QC sample were under the acceptance criteria.

Mean of post extracted spiked samples at each QC level

Matrix Effect Ratio=

Mean of aqueous samples at respective QC level

Acceptance Criteria:

Matrix effect is acceptable if the ratio is between 0.85 to 1.15 at the level of LQC, MQC and HQC.

Precision of area response of replicate injection of aqueous sample is ≤ 5% at the level of LQC, MQC and HQC.

Precision of area response of post-extracted sample is ≤ 15% at the level of LQC,MQC and HQC.

Table 5.19: Matrix Effect of Cefuroxime in Human Plasma

QC

LQC Area

MQC Area

HQC Area

Unextracted

Post Spiked

Unextracted

Post Spiked

Unextracted

Post Spiked

1

103995

89131

3180769

2480297

4471328

3278657

2

104761

87028

3184955

2489310

4468974

3321294

3

103499

86410

3182185

2580953

4250639

3415997

4

103690

86194

3144262

2586365

4251719

3431237

5

104767

88045

3329091

2614166

4243760

3423602

6

104181

88128

3334074

2641268

4301046

3440341

Mean

104149

87489.3

3225889

2565393

4331244.3

3385188

S.D (+/-)

531.86

1137.31

83233.48

66102.51

109526.11

67850.56

C.V (%)

0.51

1.30

2.58

2.58

2.53

2.00

Matrix Effect Ratio

1.19

1.26

1.28

Recovery

Recovery of an analyte in an assay is the detector response obtained from an amount of the analyte added and extracted from the biological matrix, compared to the reference standard.

Recovery was calculated by preparing QC spiking dilutions at LQC, MQC and HQC concentrations and spiking in already extracted blank samples (post-extracted sample). Concurrently, six replicates of LQC, MQC and HQC were extracted as per the extraction method (extracted sample).

Recovery of analyte and internal standard was evaluated by injecting six replicates of the extracted solutions and six replicates of the post-extracted QC samples at each LQC, MQC and HQC concentrations.

Sequence of Injection: six set of injections for each of LQC, MQC& HQC for both extracted & post-extracted sample.

Acceptance Criteria:

The recovery is deemed acceptable if the % CV ≤ 15% for both post-extracted and extracted QC samples at low, medium and high QC concentration.

Table 5.21: Recovery of Cefuroxime sodium (Analyte) from Human Plasma

QC

LQC Area

MQC Area

HQC Area

Unextracted

Extracted

Unextracted

Extracted

Unextracted

Extracted

1

83906

84026

2507596

2458741

3336542

3342111

2

85072

79160

2459727

2386457

3274075

3312426

3

82847

81088

2482737

2335346

3271147

3313607

4

83217

81499

2503586

2258623

3293427

3310967

5

85664

78017

2491656

2307580

3264442

3300864

6

84310

78742

2479333

2292168

3281242

3312236

Mean

84169.33

80422.00

2487439.17

2339819.17

3286812.50

3315368.50

S.D (+/-)

1076.43

2226.72

17553.26

72443.51

26290.29

13903.16

C.V (%)

1.28

2.77

0.71

3.10

0.80

0.42

Recovery

95.54

94.06

100.86

Overall Recovery

96.82

5.23: Recovery of Cefuroxime d3 (Internal Standard) from Human Plasma

QC

LQC Area

MQC Area

HQC Area

Unextracted

Extracted

Unextracted

Extracted

Unextracted

Extracted

1

64679

57523

64934

53938

63448

51650

2

66150

54437

64176

51512

62530

51031

3

64826

54567

65847

51909

63339

50597

4

63483

55421

65923

50506

64149

51726

5

65353

54623

64959

50814

63235

51483

6

64283

50307

63506

49998

62717

52435

Mean

64795.7

54479.7

64890.83

51446.17

63236.333

51487

S.D (+/-)

910.53

2349.01

939.67

1400.61

575.66

629.67

C.V (%)

1.41

4.31

1.45

2.72

0.91

1.22

Recovery

84.07

79.28

81.41

Overall Recovery

81.58

Stability

Standard Stock Solution Stability

Drug and IS Stock Solution Stability at Room Temperature (6 Hrs.):

Stock solution stability was carried out at 6 hours by injecting six replicates of stock dilutions of both stability standard stock solution and comparison (fresh) standard stock solution of Cefuroxime &Dihydroartemisinin. The response of stability sample was corrected by multiplying with correction factor.

Comparison Standard Stock Concentration

Correction Factor =

Stability Standard Stock Concentration

Corrected Response = Correction Factor × Stability Standard Stock Area Response

Mean Area Response of Stability Standard × Correction Factor

Mean Corrected = × 100

Response (%) Mean Area Response of Comparison Standard

Table 5.24: Stock Solution Stability of Cefuroxime at Room Temp (6 Hrs.)

Sr. No.

Stability Standard Area

Comparison Standard Area

Corrected Response

998367.0 ng/ml

1030155.8 ng/ml

1

109773

109500

113268.26

2

106226

108097

109608.32

3

108681

106348

112141.49

4

105842

104229

109212.09

5

109510

106495

112996.89

6

107667

107328

111095.20

Mean

107949.8

106999.5

111387.0

S.D (+/-)

1660.56

1784.35

1713.43

C.V (%)

1.54

1.67

1.54

Correction Factor

1.0318

Mean Response of Comparison Standard

106999.5

Mean corrected Response of Stability Standard

111387.0

% Mean Corrected Response

104.10

Table 5.26: Stock Solution Stability of IS at Room Temp (6 Hrs.)

Sr. No.

Stability Standard Area

Comparison Standard Area

Corrected Response

965185.25 ng/ml

964752.23 ng/ml

1

654308

644847

654014.5

2

644973

652585

644683.6

3

663784

632847

663486.2

4

645931

638800

645641.2

5

658164

627521

657868.7

6

640951

642768

640663.4

Mean

651351.8

639894.7

651059.6

S.D (+/-)

8808.58

8919.66

8804.63

C.V (%)

1.35

1.39

1.35

Correction Factor

1.000

Mean Response of Comparison Standard

639894.7

Mean corrected Response of Stability Standard

651059.6

% Mean Corrected Response

101.74

Acceptance Criteria:

The corrected (corrected with respect to actual concentration) mean response of the freshly prepared stock solution of analyte(s) or IS versus the comparable stored solution must be within the range of 90-110%.

Precision (% C.V) of area response of replicate injections shall be ≤ 2% in case of LC-MS/MS for both, stability stock and comparison stock.

Bench Top Stability (BTS) in Human Plasma

Bench top stability is carried to assess the stability of the analyte in biological fluids over a period of time during which the samples are expected to be kept on the bench while processing. For Bench top stability, withdraw six sets each of LQC, MQC and HQC from the deep freezer and then it was leave at room temperature for at least 6 hour. (Stability sample) This quality control samples were quantified against the freshly spiked calibration curve standards of concentration range equivalent to that used for calculation of precision and accuracy.

Table 5.27: Bench Top Stability of Cefuroxime in Human Plasma at 8.0 Hrs.

LQC(ng/ml)

MQC(ng/ml)

HQC(ng/ml)

Nominal Concentration

8.89

59.28

169.38

Acceptance Limits

7.55

10.22

50.39

68.17

143.97

194.79

Fresh

Stability

Fresh

Stability

Fresh

Stability

1

7.65

8.25

52.63

59.96

171.02

160.05

2

8.25

7.96

58.31

58.85

169.85

157.75

3

8.53

8.32

56.40

60.24

167.52

164.51

4

8.00

8.24

57.96

60.00

179.70

169.97

5

7.95

7.80

55.28

55.27

165.00

165.19

6

8.56

8.99

59.34

54.14

162.31

175.89

Mean

8.16

8.26

56.66

58.08

169.24

165.56

S.D(+/-)

0.35

0.41

2.44

2.68

6.03

6.61

C.V. (%)

4.34

4.94

4.31

4.61

3.56

3.99

% Nominal

91.78

92.94

95.57

97.97

99.91

97.74

Acceptance Criteria:

The Bench top stability evaluation is deemed acceptable if the % nominal is within ±15% compared to % nominal at 0 hour and % CV ≤ 15% at low, medium and high QC concentrations for stability sample.

At least 67% (four out of six) of total quality control sample and at least 50% quality control sample at each level should meet above acceptance criteria.

Autosampler Stability (AS)

Autosampler stability is carried out to assess the stability of the processed sample placed in the autosampler at specific temperature for the period of time depending on the anticipated run time or the complete analysis of bioanalytical batch.

In assessing the autosampler stability, six sets of QC samples (LQC, MQC and HQC) were processed and placed in the autosampler. These samples were injected after a period of 24 hours and were quantified against freshly spiked calibration curve standards of concentration range equivalent to that used for calculation of precision and accuracy.

The following result was obtained during analysis. On evaluation of results, it was concluded that % nominal and % CV were under the acceptance criteria.

Table 5.29: Autosampler Stability of Cefuroxime in Human Plasma for 24 Hrs.

LQC

MQC

HQC

Nominal Concentration(ng/ml)

8.89

59.29

169.39

Acceptance Limits

7.56

10.23

50.39

68.18

143.98

194.79

1

8.05

52.06

164.28

2

7.99

55.75

174.95

3

8.59

56.25

160.30

4

8.37

53.96

155.89

5

8.11

58.35

168.10

6

8.00

59.02

162.31

Mean

8.18

55.90

164.31

S.D(+/-)

0.24

2.62

6.61

C.V. (%)

2.98

4.69

4.02

% Nominal

92.03

94.29

97.00

Freeze-Thaw Stability (FTS)

The freeze-thaw stability is carried out to assess the stability of the analyte in biological fluids during the repeated freezing and thawing of cycles. For freeze-thaw stability, six replicates of LQC, MQC and HQC were withdrawn from deep freezer after 24 hours of continuous freezing and then thawed unassisted. Refreeze the samples after complete thawing for at least one hour. This procedure were repeat twice and then analysed on the third cycle. This Freeze-thaw quality control samples were quantified against the freshly spiked calibration curve standards of concentration range equivalent to that used for the calculation of precision and accuracy.

Table 5.31: Freeze Thaw Stability of Cefuroxime in Human Plasma

LQC

MQC

HQC

Nominal Concentration(ng/ml)

8.89

59.29

169.39

Acceptance Limits

7.56

10.23

50.39

68.18

143.98

194.79

1

7.87

51.34

155.04

2

8.37

55.58

159.84

3

8.01

54.00

152.05

4

8.55

56.99

160.55

5

8.05

54.37

145.84

6

7.93

52.03

166.31

Mean

8.13

54.05

156.61

S.D(+/-)

0.27

2.12

7.20

C.V. (%)

3.29

3.93

4.60

% Nominal

91.43

91.17

92.46

Acceptance Criteria:

The freeze and thaw stability evaluation is deemed acceptable if the % nominal is within ±15% of the nominal concentration and % CV is ≤ 15% at low, medium and high QC concentrations.

At least 67% (four out of six) of total quality control sample and at least 50% quality control sample at each level should meet above acceptance criteria.

Short Term Stability (STS) at -20°C

Stability in biological matrix at -20°C was conducted to assess the stability of analyte in case of temporary storage of plasma in -20°C deep freezer. The six sets each of LQC, MQC and HQC were stored at deep freezer after bulk spiking. Samples were withdrawn after minimum of 3 days of storage, quantifying against the freshly spiked calibration curve standards of concentration range equivalent to those used for the calculation of precision and accuracy.

Table 5.33: Short Term Stability of Cefuroxime at -20°C Storage

LQC (ng/ml)

MQC (ng/ml)

HQC (ng/ml)

Nominal Concentration

8.89

8.96

59.29

59.72

169.39

168.57

Acceptance Limits

7.56 - 10.23

7.62 - 10.30

50.39 - 68.18

50.76 - 68.68

143.98- 194.79

143.29 - 193.86

Fresh

Stability

Fresh

Stability

Fresh

Stability

1

8.64

7.99

58.66

54.22

169.96

166.86

2

8.95

8.34

54.23

58.26

172.34

165.34

3

7.83

8.82

59.03

56.36

166.2

163.85

4

8.68

8.55

55.89

52.98

164.98

167.95

5

8.64

8.92

57.34

57.75

169.34

163.02

6

8.25

8.66

56.42

57.64

165.24

166.66

Mean

8.50

8.55

56.93

56.20

168.01

165.61

S.D(+/-)

0.40

0.34

1.80

2.15

2.98

1.90

C.V. (%)

4.66

3.98

3.16

3.82

1.77

1.15

% Nominal

95.59

95.39

96.02

94.11

99.19

98.25

Correction Factor

0.994

1.013

1.014

Corrected Nominal %

99.98

100.02

99.95

Acceptance Criteria:

The short-term stability evaluation is deemed acceptable if the % corrected nominal is within ±15% of the nominal concentration and % CV ≤ 15% at low, medium and high QC concentrations.

At least 67% (four out of six) of total quality control samples and at least 50% quality control samples at each level should meet the above acceptance criteria.

Long Term Stability (LTS) at -70°C

Long-term stability evaluations are performed to check the stability of analyte at-70°C for the period of time between the date of first sample collection and the date of last sample analysis.

The stability of ARM was examined at -70°C for a period of 30 days. Six set of long term quality control samples (LQC, MQC and HQC) were withdrawn from deep freezer (-70°C) and quantified against freshly spiked calibration curve standards of concentration range equivalent to those used for the calculation of precision and accuracy.

The following result was obtained during analysis. Based on the results of analysis, it was concluded that the % nominal and % CV were under the acceptance criteria.

Long term stability should be carried out with the QC sample containing same anticoagulant used in the study samples.

Table 5.35: Long Term Stability of Cefuroxime at -70°C Storage

LQC (ng/ml)

MQC (ng/ml)

HQC (ng/ml)

Nominal Concentration

8.89

8.96

59.29

59.72

169.39

168.57

Acceptance Limits

7.56 - 10.23

7.62 - 10.30

50.39 - 68.18

50.76 - 68.68

143.98- 194.79

143.29 - 193.86

Fresh

Stability

Fresh

Stability

Fresh

Stability

1

9.86

8.51

60.27

55.22

170.05

159.56

2

9.62

8.5

58.36

57.62

167.62

160.06

3

8.26

7.99

64.08

55.31

164.34

164.32

4

8.68

8.25

58.98

51.97

166.95

155.95

5

9.26

8.62

65.59

51.85

169.82

158.63

6

8.99

8.75

59.81

54.66

167.38

156.75

Mean

9.11

8.44

61.18

54.44

167.69

159.21

S.D(+/-)

0.59

0.27

2.94

2.21

2.10

2.97

C.V. (%)

6.52

3.25

4.81

4.05

1.25

1.86

% Nominal

102.49

94.16

103.19

91.16

99.00

94.45

Correction Factor

1.080

1.124

1.053

Corrected Nominal %

100.05

100.01

99.97

Acceptance Criteria:

The long-term stability evaluation is deemed acceptable if the % corrected nominal is within ±15% of the nominal concentration and % CV is ≤ 15% at low, medium and high QC concentrations.

At least 67% (four out of six) of total quality control sample and at least 50% quality control samples at each level should meet the above acceptance criteria.