Int J App Pharm, Vol 9, Issue 1, 2017, 37-43Original Article


A UPLC-MS/MS METHOD DEVELOPMENT AND VALIDATION FOR THE ESTIMATION OF POMALIDOMIDE FROM HUMAN PLASMA

D. ATUL VASANTH*a, B. RAJKAMALb

aMewar University, Gangrar, Chittorgarh 312901, Rajasthan, India, bKVK College of Pharmacy, Surmajiguda, Hyderabad 501512, Telangana, India
Email: atulvasanth99@gmail.com

Received: 13 Oct 2016, Revised and Accepted: 05 Dec 2016


ABSTRACT

Objective: The present work aimed to develop a simple, rapid, specific and precise liquid chromatography-tandem mass spectrophotometric (LC–MS/MS) validated method for quantification of pomalidomide and internal standard (ISTD) Fluconazole in human plasma.

Methods: 50 µl of 0.1% formic acid was added to plasma samples prior to liquid-liquid extraction (LLE) using 2.5 ml of ethyl acetate. Chromatographic separation was achieved on Xterra, RP18, 5 µ (50 x 4.6 mm) column using a mixture of 0.1% (v/v) formic acid in water to methanol at a ratio of 12:88, v/v as the mobile phase. The flow rate was 0.50 ml/min. The LC eluent was split, and approximately 0.1 ml/min was introduced into Tandem mass spectrometer using turbo Ion Spray interface at 325 °C. Quantitation was performed by transitions of m/z 260.1 precursor ion to the m/z 148.8 for pomalidomide and m/z 307.1/238.0 for fluconazole.

Results: The concentrations of nine working standards showed linearity between 9.998 to 1009.650 ng/ml (r2 ≥ 0.9968). Chromatographic separation was achieved within 2 min. The average extraction recoveries of three quality control concentrations were 53.86% for pomalidomide and were within the acceptance limits. The coefficient of variation was ≤15% for intra-and inter-batch assays. The %CV of ruggedness ranges 1.32 to 4.03. The % stability of short term and long term stock solution stability studies was found to be 99.01% and 98.49% respectively.

Conclusion: The results obtained for specificity, linearity, accuracy, precision, ruggedness and stability studies were within limits. Thus the validated economical method was applied for pharmacokinetic studies of pomalidomide.

Keywords: Pomalidomide, LC-MS/MS, Human plasma, Liquid-liquid extraction


INTRODUCTION

Pomalidomide chemically 4-amino-2-(2, 6-dioxopiperidin-3-yl) isoindoline-1, 3-dione, the newest immune-modulatory drugs (IMiD), was designed to be more potent and less toxic than thalidomide and lenalidomide [1-2]. It is used for the treatment of relapsed and refractory multiple myeloma. Dr. Rober D'Amato's labs led to the first report [3-4] stating that 3-amino-thalidomide was able to directly inhibit both the tumor cell and vascular compartments of myeloma cancers. An HPLC-UV method was reported for inversion of pomalidomide in phosphate-buffered saline and human plasma in μg/ml range [5]. The LC-MS methods were also published for the Pharmacokinetic study of pomalidomide but no validation details were presented [6-7]. A validated UPLC-MS method was reported using negative ionisation mode for determination of pomalidomide from rat plasma [8], Described here is a simple, sensitive, and selective LC-MS/MS method for pomalidomide in the human plasma concentration range of 9.998 to 1009.650 ng/ml. As there is no literature on stability data of pomalidomide in human plasma, this study performed assay validations, according to the FDA guidelines [9]. While this method with validation details were economical and applied for pharmacokinetic studies of pomalidomide.

MATERIALS AND METHODS [8]

Apparatus and software

The HPLC pump (Agilent 1200 series Binary SL) with an autosampler (Agilent 1200 series Hip-ALSSL) was coupled with Agilent 6460 Triple Quad Tandem mass spectrometer. The column oven was Agilent 1200 series TCC SL. The chromatographic integration was performed by Agilent mass hunter software.

Chemicals and reagents

Pomalidomide and Fluconazole (IS) were procured from NATCO Pharma Ltd., Hyderabad, Formic acid, Methanol and ethyl acetate was procured from Merck Specialities Pvt. Ltd, Mumbai, India. Water used was collected from water purification systems (Milli Q, Milli Pore, USA) installed in the laboratory. Pooled drug-free expired frozen human plasma (K2-EDTA as anticoagulant) was obtained froma Blood Bank, Hyderabad, was used during validation and study sample analysis. The plasma was stored into-70±5 °C.

Standards and working solutions

Calibration standard solutions

Stock solutions of pomalidomide and Fluconazole internal standard (IS) were prepared in methanol. Further dilutions were carried out in 50% methanol. Calibration standards of nine concentration levels were prepared freshly by spiking drug-free plasma with pomalidomide stock solution to give the concentrations of 9.998, 25.241, 50.281, 150.438, 301.885, 503.815, 705.745, 906.666 and 1009.650 ng/ml.

Quality control standards

Lowest quality control standards, Median quality control standards and highest quality control standards were prepared by spiking drug-free plasma with pomalidomide to give a solution containing 26.248, 323.056 and 807.640 ng/ml respectively. They were stored at-20 °c till the time analysed.

Chromatographic conditions

Chromatographic separation was performed on Xterra, RP18, 5 µ (50 x 4.6 mm), analytical column and the mobile phase was a mixture of 0.1% (v/v) formic acid in water to methanol at a ratio of 12:88, v/v. Injection volume was 10μL. The flow rate was 0.50 ml/min. Total analysis time of single injection was 2.0 min. Column oven temperature and autosampler temperature was set to 30 °C and 10 °C, respectively.

Mass spectrometric conditions

The LC eluent was split, and approximately 0.100 ml/min was introduced via electrospray ionisation using a Turbo Ion Spray interface set at 325 °C to generate positive ions [M+H]+. The Mass spectrometric parameters were optimised as shown in table no 1.

Table 1: Mass spectrometric conditions

Capillary voltage 3500V
Nozzle voltage 1500V
Delta EMV(+) 500 Positive
Gas flow 5 L/min
Gas temperature 350 °C
Nebulizer pressure 25 psi
Sheath gas temperature 300 °C
Sheath gas flow 11L/min
Acquisition
Parameters Pomalidomide
Transition 260.1/148.8 (m/z)
Polarity Positive
MS1 resolution Unit
MS2 resolution Unit
Dwell time (millisec) 200
Fragmentor (V) 100
Collision energy (V) 8

Sample preparation method

To 250 µl of plasma, 50 µl of ISTD (1µg/ml) and 50 µl of 0.1% formic acid was added and vortexed. The drug was extracted with 2.5 ml of ethyl acetate, followed by centrifugation at 2000 rpm/min on a cooling centrifuge for 15 min at 4 °C. The supernatant of 2 ml was withdrawn and evaporated at 50 °C 15 psi of nitrogen until dryness at LV evaporator. The residue was reconstituted with 500 µl of mobile phase and respective samples were injected into the column.

Validation

Specificity

A solution containing 9.999 ng/ml was injected onto the column under optimised chromatographic conditions to show the separation of pomalidomide from impurities and plasma. The specificity of the method was checked for the interference from plasma.

Linearity

Spiked concentrations were plotted against peak area ratios of pomalidomide to the internal standard, and the best fit line was calculated. Wide range calibration was determined by solutions containing9.998 to 1009.650ng/ml.

Recovery studies

The % mean recoveries were determined by measuring the responses of the extracted plasma Quality control samples at HQC, MQC and LQC against un-extracted Quality control samples at HQC, MQC and LQC.

Precision and accuracy

The between-run (Inter-day) accuracy and precision evaluation were assessed by the repeated analysis of human K3 EDTA plasma samples containing different concentrations of pomalidomide on separate occasions. A single run consisted of a calibration curve plus six replicates of the lower limit of quantitation, low, medium and high-quality control samples.

Within-run (Intraday) accuracy and precision evaluations were performed by analysing replicate concentrations of pomalidomide in human K3 EDTA plasma. The run consisted of a calibration curve plus a total of 24 spiked samples, six replicates of each of the LLOQ, lower, medium and higher quality control samples.

Matrix effect

Blank plasma samples of 6 different human K3 EDTA plasma sources were processed and spiked with aqueous low-quality control and high-quality control (post extraction addition) and analysed in a single run along with diluted pure standard at each concentration level.

Ruggedness

The ruggedness of the method was assessed by analysing a precision and accuracy batch using a different column, by the different analyst in another instrument.

Stability studies

Short-term stock solution stability of pomalidomide

Solutions of pomalidomide were prepared in methanol (Stability Samples) and were kept at room temperature for 6 h 30 min. A freshly prepared solution of pomalidomide (Comparison Samples) and stability samples were diluted at approximately the same analyte concentration and analysed in a single run; analyte responses were used to determine % stability over time.

Short-term stock solution stability of internal standard

Solutions of internal standard (Fluconazole) were prepared in methanol (Stability Samples) and were kept at room temperature for 6 h 30 min. A freshly prepared solution of internal standard (Comparison Samples) and stability samples were diluted at approximately the same analyte concentration and analyzed in a single run; Analyte responses were used to determine % stability over time.

Freeze-thaw stability

Samples were prepared at low and high-quality control levels, aliquoted and frozen at-70 °C. Some of the aliquots of quality control samples were subjected to five freeze-thaw cycles (stability samples). A calibration curve and quality control samples were freshly prepared (Comparison Samples) and processed with 6 replicates of stability samples and analysed in a single run.

Long-term stock solution stability of pomalidomide

Solutions of Pomalidomide were prepared in methanol (Stability Samples) and were kept at refrigerator (2-8 °C) for 10 D 02 H. A freshly prepared solution of pomalidomide (Comparison Samples) and stability samples were diluted at approximately the same analyte concentration and analysed in a single run.

Long-term stock solution stability of internal standard

Solutions of Internal standard were prepared in methanol (Stability Samples) and were kept at refrigerator (2-8 °C) for 10 D 02 H. A freshly prepared solution of internal standard (Comparison Samples) and stability samples were diluted at approximately the same analyte concentration and analysed in a single run

RESULTS AND DISCUSSION

The chromatography observed during the course of validation was acceptable and representative chromatograms of, LLOQ, LQC, MQC, HQC, internal standard (ISTD) and standard blank samples are shown in (fig. 1).

The method developed was validated for specificity, accuracy and precision, linearity, ruggedness and stability as per USFDA guidance [10-12]. The results of validating parameters are given below.

Specificity

Nine different lots of plasma were analysed to ensure that no endogenous interferences were present at the retention time of pomalidomide and fluconazole. Nine LLOQ (9.999 ng/ml) level samples along with plasma blank from the respective plasma lots were prepared and analysed. (table 2) shows results of specificity. In all plasma blanks, the response at the retention time of pomalidomide was less than 20% of LLOQ response, and at the retention time of IS, the response was less than 5% of mean IS response in LLOQ. The typical chromatogram of plasma blank and chromatogram of LLOQ was shown in (fig. 1).

Fig. 1: Chromatograms of LLOQ, quality control samples, ISTD and blank matrix


Table 2: Results of specificity for pomalidomide and fluconazole (ISTD)

 

Analyte

IS

Area of interfering peak at RT of analyte

Area observed for extracted LLOQ

% Interference at RT of analyte

Area of interfering Peak at RT of ISTD

Area observed for extracted ISTD

% Interference at RT of ISTD

01

0

15286

0

0

575244

0

02

0

13288

0

0

582214

0

03

0

11110

0

0

578922

0

04

0

11440

0

0

564562

0

05

0

11402

0

0

558925

0

06

0

11059

0

0

589001

0

07

0

11215

0

0

591285

0

08

0

11580

0

0

578862

0

09

0

12089

0

0

588638

0

Mean

12052.111

MEAN

180201.89


Linearity

The calibration curve (peak area ratio Vs Concentration) was linear over working a range of 9.998 to 1009.650 ng/ml with nine point calibration used for quantification by linear regression, shown in (fig. 2). The regression equation for the analysis was Y=0.0039x-0.0089 with coefficient of correction (r2) = 0.99686.

Fig. 3: Spiked concentrations (9.998 to 1009.650 ng/ml) were plotted against calculated concentration Vs concentration with ten point calibration used for quantification by linear regression

Recovery

The % mean recovery for pomalidomide in LQC, MQC and HQC was 50.68%, 53.43% and 57.49% respectively (table 3).

Table 3:% Mean recovery of pomalidomide for LQC, MQC and HQC

LQC 26.248ng/ml

MQC 323.056 ng/ml

HQC 807.640 ng/ml

Aqueous analyte response

Extracted analyte response

Aqueous analyte response

Extracted analyte response

Aqueous analyte response

Extracted analyte response

01

240986

49902

2785844

599122

6694089

1549621

02

245029

49079

2784946

595935

6765199

1550584

03

244894

48906

2792062

593232

6782112

1564921

04

243537

48828

2785487

591894

6835789

1571431

05

240892

49190

2779585

593165

6838061

1568923

06

238416

48789

2785210

598329

6870744

1573663

Mean

242292.3

49115.6

2785522.3

595279.5

6797665.6

1563190.5

SD (+)

2626.8

414.3

3963.6

2987.2

63952.7

10549.7

CV (%)

1.08

0.84

0.14

0.50

0.94

0.67

Conc. Factor

250

250

250

Mean Recovery

50.68

53.43

57.49

Global Recovery

53.86


Intraday (within run) and inter-day (between run) precision and accuracy

The within-run coefficients of variation ranged between 1.47% and 4.57% for pomalidomide. The within-run percentages of nominal concentrations ranged between 97.95% and 107.58% for pomalidomide. Results are presented in table 4.

The between-run coefficients of variation ranged between 2.88% and 4.22% for pomalidomide. The between-run percentages of nominal concentrations ranged between 99.41% and 106.97% for pomalidomide. Results are presented in table 5.

Matrix effect

The percentage matrix effect of analyte was found to be-0.25 and 2.74 for pomalidomide for low and high-quality control samples. Results are presented in table 6.

Ruggedness

The coefficients of variation ranged between 1.32% and 4.03% for pomalidomide. The percentages of nominal concentrations ranged between 101.06% and 110.08% for pomalidomide. Results are presented in table 7.

Table 4: Intraday precision and accuracy of quality control standard

 S. N0.

LLOQ 9.999 ng/ml

LQC 26.248ng/ml

MQC  323.056 ng/ml

HQC 807.640 ng/ml

Conc. found (ng/ml)

% nominal Conc

Conc. found (ng/ml)

% nomi Conc nal

Conc. found (ng/ml)

% nominal Conc

Conc. found (ng/ml)

% nominal Conc

 1

10.705

107.06

28.081

106.98

315.669

97.71

762.368

94.39

 2

10.569

105.70

27.895

106.27

355.681

110.10

782.178

96.85

 3

10.411

104.12

27.857

106.13

318.659

98.64

775.543

96.03

 4

11.487

114.88

27.942

106.45

324.372

100.41

810.934

100.41

 5

10.61

106.11

27.841

106.07

317.699

98.34

805.069

99.68

 6

10.758

107.60

28.915

110.16

327.842

101.48

810.606

100.37

N

6

6

6

6

6

6

6

6

Mean

10.757

107.58

28.089

107.01

326.654

101.11

791.116

97.95

SD(±)

0.38

 

0.41

 

14.93

 

20.57

 

CV(%)

3.51

1.47

4.57

2.60


Table 5: Inter day precision and accuracy of quality control standard

Batch ID

LLOQ 9.999 ng/ml

LQC 26.248ng/ml

MQC 323.056 ng/ml

HQC 807.640ng/ml

Conc. found (ng/ml)

% nominal conc

Conc. found (ng/ml)

% nominal conc

Conc. found (ng/ml)

% nominal conc

Conc. found (ng/ml)

% nominal conc

10.705

107.06

28.081

106.98

315.669

97.71

762.368

94.39

10.569

105.70

27.895

106.27

355.681

110.10

782.178

96.85

PandA-01

10.411

104.12

27.857

106.13

318.659

98.64

775.543

96.03

11.487

114.88

27.942

106.45

324.372

100.41

810.934

100.41

10.610

106.11

27.841

106.07

317.699

98.34

805.069

99.68

10.758

107.60

28.915

110.16

327.842

101.48

810.606

100.37

10.322

103.23

27.867

106.17

311.270

96.35

762.021

94.35

10.385

103.86

26.741

101.88

355.184

109.95

783.091

96.96

PandA-02

10.609

106.10

26.569

101.22

315.344

97.61

762.157

94.37

10.896

108.97

27.498

104.76

312.676

96.79

798.395

98.86

10.268

102.69

27.581

105.08

313.590

97.07

786.741

97.41

10.307

103.08

27.235

103.76

317.332

98.23

793.101

98.20

10.729

107.30

28.810

109.76

330.628

102.34

775.171

95.98

10.867

108.69

28.880

110.03

318.223

98.50

838.267

103.79

PandA-03

10.309

103.10

29.561

112.62

326.177

100.97

830.304

102.81

10.321

103.22

29.126

110.97

328.816

101.78

874.482

108.28

10.805

108.06

29.018

110.55

327.572

101.40

854.598

105.81

10.443

104.44

27.968

106.55

327.423

101.35

846.592

104.82

N:

18

18

18

18

18

18

18

18

Mean:

10.600

106.01

28.077

106.97

324.675

100.50

802.868

99.41

SD(±):

0.31

0.83

12.76

33.89

CV (%):

2.88

2.95

3.93

4.22


Table 6: Results of matrix effect obtained by preparing LQC and HQC with six different lots of plasma

S. No. AQS LQC response PEX LQC response AQS HQC response PEX HQC response
1 195688 203204 5680638 5635662
2 200736 197892 5694436 5694225
3 200626 197423 5637040 6355785
4 198973 198785 5647571 5716531
5 200778 199084 5680208 5753628
6 201077 198505 5655245 5772353
Mean 199646.333 199148.833 5665856.333 5821364
SD 2078.29 2076.23 22443.71 266171.21
%CV 1.04 1.04 0.4 4.57
%ME -0.25   2.74  

Table 7: Results of ruggedness

S. No.

LLOQ 9.999 ng/ml

LQC 26.248ng/ml

MQC 323.056 ng/ml

HQC 807.640 ng/ml

Conc. found (ng/ml)

% nominal conc

Conc. found (ng/ml)

%
nominal  conc

Conc. found (ng/ml)

% nominal conc

Conc. found (ng/ml)

% nominal conc

 1

10.729

107.30

28.810

109.76

330.62

102.34

775.171

95.98

 2

10.867

108.69

28.880

110.03

318.22

98.50

838.267

103.79

 3

10.309

103.10

29.561

112.62

326.17

100.97

830.304

102.81

 4

10.321

103.22

29.126

110.97

328.81

101.78

874.482

108.28

 5

10.805

108.06

29.018

110.55

327.57

101.40

854.598

105.81

 6

10.443

104.44

27.968

106.55

327.42

101.35

846.592

104.82

N

6

6

6

6

6

6

6

6

Mean

10.579

105.80

28.894

110.08

326.47

101.06

836.569

103.58

SD(±)

0.25

 

0.53

 

4.31

 

33.69

CV (%)

2.37

1.82

1.32

4.03

Stability studies

Short-term stock solution stability of pomalidomide

Pomalidomide is found to be stable in methanol for 6 h 30 min at room temperature with a % stability of 99.01%. Results are presented in table 8.

Short-term stock solution stability of internal standard

The internal standard is found to be stable in methanol for 6 h 30 min at room temperature with a % stability of 99.15%. Results are presented in table 9.

Freeze-thaw stability

Pomalidomide is found to be stable in human K3 EDTA plasma after five freeze-thaw cycles at-70 °C with coefficients of variation of 3.27% (LQC) and 3.86% (HQC) for pomalidomide, and the percentages of nominal concentrations for pomalidomide were found to be 103.17% (LQC) and 101.23% (HQC). Results are presented in table 10.

Long-term stock solution stability of pomalidomide

Pomalidomide is found to be stable in methanol 10 D 02 H at refrigerator (2-8 °C) with a % stability of 98.49% for pomalidomide. Results are presented in table 11.

Long-term stock solution stability of internal standard

The internal standard is found to be stable in methanol 10 D 02 H at refrigerator (2-8 °C) with a % stability of 97.74%. Results are presented in table 12.

Table 8: Short-term stock solution stability of analyte

S. No.

Analyte

SS

CS

1

2564444

2606664

2

2597482

2616699

3

2606795

2630379

4

2611068

2627041

5

2598633

2630048

6

2608998

2629280

Mean

2597903.333

2623351.833

SD

17295.66

9651.08

%CV

0.67

0.37

% stability

99.01


Table 9: Short-term stock solution stability of internal standard

S. No.

ISTD

Stability solution

Comparison solution

1

2504198

2521525

2

2510082

2524682

3

2498215

2537401

4

2487925

2519696

5

2512367

2504040

6

2491004

2526432

Mean

2500631.833

2522296.000

SD

9997.37

10877.47

%CV

0.40

0.43

% stability

99.15


Table 10: Freeze-thaw stability at-70 °C

S. No.

Freshly spiked

Freeze-thaw

LQC

HQC

LQC

HQC

Nominal Con (ng/ml)

Nominal Con (ng/ml)

26.249

807.648

26.248

807.64

1

28.019

791.749

29.380

801.458

2

27.816

767.862

30.051

839.017

3

27.327

789.495

28.055

824.962

4

27.830

821.854

27.867

754.842

5

27.819

842.104

28.198

791.864

6

27.272

770.159

27.791

830.043

Mean

27.680

797.204

28.557

807.031

SD

0.31

29.34

0.93

31.17

%CV

1.10

3.68

3.27

3.86

% stability

103.17

101.23


Table 11: Long-term stock solution stability of analyte

S. No.

Analyte

SS

CS

1

2280682

2314048

2

2287758

2311173

3

2291699

2307848

4

2288329

2312386

5

2300006

2324564

6

2287308

2353235

Mean

2280682

2314048

SD

6355.03

16985.98

%CV

0.28

0.73

% stability

98.49


Table 12: Long-term stock solution stability of internal standard

S. No.

ISTD

Stability solution

Comparison solution

1

2458845

2520014

2

2448795

2531462

3

2431526

2512650

4

2484415

2526423

5

2478462

2513284

6

2460025

2500186

Mean

2460344.7

2517336.5

SD

19359.133

11151.5822

%CV

0.79

0.44

% stability

97.74


CONCLUSION

Chromatographic separation was achieved on Xterra, RP18, 5 µ (50 x 4.6 mm) column using a mixture of 0.1% (v/v) formic acid in water to methanol at a ratio of 12:88, v/v as the mobile phase. The drug was extracted with 2.5 ml of ethyl acetate. The specificity of the method was checked for the interference from plasma. The calibration curve (peak area ratio Vs Concentration) was linear over working a range of 9.998 to 1009.650 ng/ml with nine point calibration used for quantification by linear regression. The % mean recovery for pomalidomide in LQC, MQC and HQC was 50.68%, 53.43% and 57.49% respectively. The within-run coefficients of variation ranged between 1.47% and 4.57% for pomalidomide. The between-run coefficients of variation ranged between 2.88% and 4.22% for pomalidomide. The percentage matrix effect of analyte was found to be-0.25 and 2.74 for pomalidomide for low and high-quality control samples. The stability test was performed to assess the long term and short term stability of sofosbuvir sample solutions, internal standard solutions. The developed method was validated for the quantitative determination of sofosbuvir from plasma was simple, rapid, specific, sensitive, accurate and precise. Hence, the method is quite suitable to detect the drug from plasma samples of human volunteers.

ACKNOWLEDGEMENT

I am also grateful to my scholars and my friends for their kind help from time to time at each and every step of my project work.

CONFLICT OF INTERESTS

Declared none

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How to cite this article

  • D Atul Vasanth, B Rajkamal. A UPLC-MS/MS method development and validation for the estimation of pomalidomide from human plasma. Int J Appl Pharm 2017;9(1):37-43.


About this article

Title

A UPLC-MS/MS METHOD DEVELOPMENT AND VALIDATION FOR THE ESTIMATION OF POMALIDOMIDE FROM HUMAN PLASMA

Keywords

Pomalidomide, LC-MS/MS, Human plasma, Liquid-liquid extraction

DOI

10.22159/ijap.2017v9i1.15653

Date

31-12-2016

Additional Links

Manuscript Submission

Journal

International Journal of Applied Pharmaceutics
Vol 9, Issue 1, 2017 Page: 37-43

Online ISSN

0975-7058

Statistics

51 Views | Downloads

Authors & Affiliations

D. Atul Vasanth
Mewar University, Gangrar, Chittorgarh 312901, Rajasthan, India
India

B. Rajkamal
KVK college of pharmacy, Surmajiguda, Hyderabad-501512, Telangana, India
India


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