Chapter 3 - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/5681/11/11...Injection (4th generation cephalosporin antibiotic)were used for determination of NID by gel clot technique

36

Chapter 3

Detection of NID Factor in Cephalosporin Antibiotics

37

3.1 Detection of NID Factor in 1st to 4th Generation Cephalosporin Antibiotics Introduction:

Cephalosporin antibiotics are a broad class of antibiotics. They are the most

widely used life saving drugs. The bacterial Endotoxins test to be passed as

per the respective pharmacopoeial monograph before releasing the drug

into market57 but due to interfering factors in most of the cases there is a

probability of getting false positive results. So Non-interfering dilution (NID) to

be selected before validating a product. Single product from 1st to 4th

Generation cephalosporin’s were selected for study and the NID factor

among those Cephalosporin antibiotics was between + two fold. It was

concluded that NID among different molecules of Cephalosporin antibiotics

was mostly + two fold.

Materials and Methods:

Materials:

Lyophilized Limulus Amoebocyte Lysate of 0.125sensitivity (LAL), Control

Standard Endotoxin 5 Eu/ng (CSE), LAL Reagent Water (LRW) of Endosafe US,

Depyrogenated (250°C for 30 min) 10 X 75 mm assay tubes, 16X100 mm

dilution tubes, pyrogen free Micropipette tips, vortex mixture, 1N NaoH, 1N

Hcl, Cephalothin for Injection (1st generation cephalosporin antibiotic),

Cefuroxime Sodium (2nd generation cephalosporin antibiotic), Ceftriaxone

Sodium (3rd generation cephalosporin antibiotic) and Cefpirome sulphate for

Injection (4th generation cephalosporin antibiotic)were used for determination

of NID by gel clot technique.

The sensitivity of the Lysate (labeled 0.125Eu/mL) was determined by

using known amount of E.coli Control Standard Endotoxin.

In the gel-clot techniques, the reaction end point is determined from dilutions

of the material under test in direct comparison with parallel dilutions or a

reference Endotoxin, and quantities of Endotoxins are expressed in Endotoxin

units.

3.1 Detection of NID Factor in 1st to 4th Generation Cephalosporin Antibiotics

38

1. Preparation of Standard stock solution and standard solutions: The CSE

having a defined potency of 50 EU/Vial was reconstituted with 5ml of LRW

and mixed intermittently for 30 minutes using a vortex mixture and this

concentrate was used to prepare 2λ, λ, λ/2 & λ/4, where λ is the labeled

claim sensitivity of Lysate.

2. Preparation of sample solution: Test samples were diluted to the required

concentrations based on the formulae MVD. MVD is the maximum valid

dilution, which is allowable dilution of the specimen at which the Endotoxin

limit can be determined. The general equation to determine MVD is

MVD = (Endotoxin limit X Concentration of sample solution)/ (λ).

Where E.L is the Endotoxin limit of the test sample, which is specified in the

individual monograph in terms of volume or units of active drug (in EU/mg).

3. Cephalothin for Injection sample preparation: Batch No: CFI-0109,

Potency=50 mg/mL , E.L=0.13 Eu/mg , Lysate sensitivity is 0.125 Eu/mL and

MVD = 52.The following test dilutions are prepared by 1:52 (0.96 mg/mL), 1:26

(1.92 mg/mL), 1:13 (3.84 mg/mL), 1:6.5 (7.69 mg/mL) & 1:3.2 (16.38 mg/mL).

4. Cefuroxime Sodium sample preparation: Batch No: CXN-0109, Potency=50

mg/mL , E.L=0.10 Eu/mg , Lysate sensitivity is 0.125 Eu/mL and MVD = 40.The

following test dilutions are prepared by 1:40 (1.25 mg/mL), 1:20 (2.5 mg/mL),

1:10 (5 mg/mL), 1:5 (10 mg/mL) & 1:2.5 (20 mg/mL).

5. Ceftriaxone Sodium sample preparation: Batch No: CTN-0109, Potency=50

mg/mL, E.L=0.20 Eu/mg, Lysate sensitivity is 0.125 Eu/mL and MVD = 80. The


1:20 (2.5 mg/mL), 1:10 (5 mg/mL) & 1:5 (10 mg/mL).

6. Cefpirome sulphate for Injection sample preparation: Batch No: CSI-0109,

Potency=50 mg/mL, E.L=0.20 Eu/mg, Lysate sensitivity is 0.125 Eu/mL and MVD

= 80. The following test dilutions are prepared by 1:80 (0.62 mg/mL), 1:40 (1.25

mg/mL), 1:20 (2.5 mg/mL), 1:10 (5 mg/mL) & 1:5 (10 mg/mL).


39

Above mentioned Cephalosporin antibiotics are validated after adjusting

their pH to 6-8, The NID factor was evaluated in the preliminary screening,

variation among them is + two fold.

Methods

Equal volume of test sample and LAL reagent is added in a

depyrogenated test tube of 10 X 75 mm and incubate this mixture at 37± 1°C

for 60±2 min. Then invert the tube by 180° and look for gel formation. If a gel

inside the test tube is able to maintain its integrity after inverting the tube to

180° then it is a positive reaction which indicates presence of Endotoxin in the

sample greater than the limit. Other than this any condition is considered as

negative which indicates absence of Endotoxin in the sample (lesser than the

lysate sensitivity).

Product Testing: For testing products equal volume of drug (sample) and LAL

reagent is taken and following tubes are prepared (USP 32, 2009)

Negative Product Control (NPC) - Sample + LAL

Positive Product Control (PPC) - Sample + CSE (2λ) + LAL

Negative Water Control (NWC) - LRW + LAL

Positive Water Control (PWC) - LRW + CSE (2λ) + LAL

Majority of times it has been a common observation that if a product is tested

directly it inhibits the LAL test and thus shows interference23 (van Noordwijk et

al., 1997).

Interference: Interference is defined as a significant difference between the

end points of positive water control and positive product control using

standard Endotoxin.

This interference could be either inhibition wherein the recovery of Endotoxin

is lower than the expected or enhancement wherein the recovery of

Endotoxin is higher than expected.

Product Validation: Product needs to be validated before start for routine

testing. Validation is a test condition where an Endotoxin standard is

detected with the same efficiency in a test sample as it is in LRW. This

validation study consists of two different phases wherein in Phase I (Preliminary

screening) involve interference testing and Phase II consists of validation of

product.


40

Significance of product validation is that it gives information on whether there

are any interfering factors in the drug product to the LAL test and also it gives

an idea of the approximate levels of Endotoxin content in the drug product. It

also covers manufacturing of product and formulation of the product.

It is always advisable to carry out revalidation if product formulation is

changed and which is likely to affect the interference pattern of the product

for LAL test. Also revalidation is to be conducted for any product if there is any

change in manufacturing procedures or vendor.

Results and discussions:

Phase I: Preliminary Screening / interference Study25 (Cooper, 1990).

In this two identical series of product dilutions (two-fold dilutions), one

spiked with 2λ, and one left unspiked. The result of Phase I will tell you the

non-interfering dilution (NID) of the product. The non-interfering dilution (NID)

is the first set of PPC that shows a gel.

Cephalothin for Injection:1st Generation Cephalosporin) NID-1:6.5 (1:13 selected for validation)

Sample Dilution 1:3.2 1:6.5 1:13 1:26 1:52 Unspiked -- ++ -- -- --

Spiked -- ++ ++ ++ ++ Cefuroxime Sodium: (2nd Generation Cephalosporin) NID-1:2.5 (1:10 selected for validation)

Sample Dilution 1:2.5 1:5 1:10 1:20 1:40 Unspiked ++ ++ -- -- --

Spiked ++ ++ ++ ++ ++ Ceftriaxone Sodium: (3rd Generation Cephalosporin) NID-1:5 (1:20 selected for validation)

Sample Dilution 1:5 1:10 1:20 1:40 1:80 Unspiked ++ ++ -- -- --

Spiked ++ ++ ++ ++ ++ Cefpirome sulphate for Injection: (4th Generation Cephalosporin) NID-10(1:40 selected for validation)

Sample Dilution 1:5 1:10 1:20 1:40 1:80 Unspiked -- ++ ++ -- --

Spiked -- ++ ++ ++ ++ Table: 1 Results of NID factor in 1st to 4th Generation Cephalosporin’s; -- No spike recovery


41

Assay results after preliminary screening with pH adjusting to 6-8 are presented

in Table 1.

This assay shows no inhibition from 1:6.5 dilution onwards in Cephalothin for

Injection, 1:2.5 in Cefuroxime Sodium, 1:5 in Ceftriaxone Sodium and 1:10 in

Cefpirome sulphate for Injection and the spike recovery from the NID

onwards. It is advisable to validate the product next to MVD to take care of

any batch to batch variation during regular production in the

pharmaceutical industries.

Phase II: Validation of Product

For validation, test and compare two identical series of Endotoxin dilutions

bracketingλ; One prepared in LRW and another prepared in product diluted

to the proposed test dilution. Refer table 1 for dilution selected for validation

(Hot spike method).

Phase: II Results:

Cephalothin for Injection Cefuroxime Sodium

Repl

icat

es

0.

25

Eu/m

L

0.

125

Eu/m

L

0.

0625

Eu

/mL

0.

0312

Eu

/mL

0.

25

Eu/m

L

0.

125

Eu/m

L

0.

0625

Eu

/mL

0.

0312

Eu

/mL

1 + + - - + + - - 2 + + - - + + - - 3 + + - - + + - - 4 + + - - + + - -

Ceftriaxone Sodium Cefpirome sulphate for Injection

Repl

ica

tes

0.

25

Eu/m

L

0.

125

Eu/m

L

0.

0625

Eu

/mL

0.

0312

Eu

/mL

0.

25

Eu/m

L

0.

125

Eu/m

L

0.

0625

Eu

/mL

0.

0312

Eu

/mL

1 + + - - + + - - 2 + + - - + + - - 3 + + - - + + - - 4 + + - - + + - -

Endotoxin/product; Negative product control: --; Geometric Mean = 0.125 EU/ml. Table: 2

Assay results of the products after spiking with known concentration of

Endotoxin are presented in Table 2.


42

Replicates 0.25 Eu/mL 0.125 Eu/mL 0.0625 Eu/mL 0.0312 Eu/mL 1 + + - - 2 + + - - 3 + + - - 4 + + - -

Table: 3 Endotoxin/LRW; Negative product control: --; Geometric Mean = 0.125 EU/ml. Assay results of label claim sensitivity of the lysate are presented in Table 3.

Successful validation requires that both series confirm label claim (Geometric

mean) within +/- one two-fold dilution. Validation is conducted at this dilution

on three batches of product.

Figure 1: Cephalothin for Injection (1st Generation)

Figure 2: Cefuroxime Sodium (2nd Generation)


43

Figure 3: Ceftriaxone Sodium (3rd Generation)

Figure 4: Cefpirome sulphate for Injection (4th Generation)

Discussion:

Analytical results of the Cephalosporin group from table 1 after pH

adjustment reveal that NID factor between the four products is between

MVD/8 and MVD/16. In table 2 results shows 100% recovery of the spiked

Endotoxins into the product. It means there is no inhibition or enhancement in

these drug products during quantification of Endotoxins, So there is no

possibility of false positive results and we can apply the same methodology to

understand the behavior of various cephalosporin group drug products

before proceeding for actual testing. It was concluded that NID among four

generations of Cephalosporin antibiotics was mostly + two fold.

44

3.2 Resolving Test Interference In Detection of Endotoxin’s in 3rd Generation

Cephalosporin Drug:

Introduction:

The effect of varying the pH and ionic strength in the raw materials used in

the finished product, 3rd generation Cephalosporin drug Cefepime for

Injection was investigated. The interfering factors inhibiting the Endotoxins in

the raw material Cefepime hydrochloride and L-Arginine while quantifying

with Limulus Amoebocyte Lysate are sorted out by neutralizing the ionic

concentration in the raw materials using dilute alkali/ acid and validated the

final product along with its raw materials. This technique can be used for

other cephalosporin group and parenteral drugs to overcome the

interference.


Lyophilized Limulus Amoebocyte Lysate of 0.125 sensitivity (LAL), Control

Standard Endotoxin 5 Eu/ng CSE), LAL Reagent Water of Endosafe US,

Depyrogenated (250°C for 30 min )10 X 75 mm assay tubes, 16X100 mm

dilution tubes ,pyrogen free Micropipette tips, vortex mixture, 1N NaoH, 1N

Hcl, Cefepime Hcl (Sterile), L-Arginine (Sterile) and Cefepime for Injection

were used for determination of Endotoxin content by the gel clot technique.

The sensitivity of the Lysate (labeled 0.125 Eu/mL) was determined by using

known amount of E.coli Control Standard Endotoxin.




units.










3.2 Resolving Test Interference In Detection Of Endotoxin’s In 3rd Generation Cephalosporin Drug

45

MVD = (Endotoxin limit X Concentration of sample solution)/ (λ). Where E.L is

the Endotoxin limit of the test sample, which is specified in the individual

monograph in terms of volume or units of active drug (in EU/mg).

3. Cefepime Hcl sterile sample preparation: Batch No: CHS-0101,

Potency=100mg/mL, E.L=0.04 Eu/mg, Lysate sensitivity is 0.125 Eu/mL and MVD

= 32. The following test dilutions are prepared by 1:32 (3.125 mg/mL), 1:16

(6.25 mg/mL), 1:8 (12.5 mg/mL), 1:4 (25 mg/mL) & 1:2 (50 mg/mL).

4. L-Arginine sterile sample preparation: Batch No:LAS-0102,

Potency=100mg/mL, E.L=0.03 Eu/mg , Lysate sensitivity is 0.125 Eu/mL and

MVD = 24.The following test dilutions are prepared by 1:24 (4.16 mg/mL), 1:12

(8.33 mg/mL), 1:6 (16.66 mg/mL), 1:3 (33.33 mg/mL) & 1:1.5 (66.66 mg/mL)

5. Cefepime for Injection sample preparation: Batch CPI -0103,

Potency=100mg/mL , E.L=0.06 Eu/mg , Lysate sensitivity is 0.125 Eu/mL and

MVD = 48. The following test dilutions are prepared by 1:48 (2.08 mg/mL), 1:24

(4.16 mg/mL), 1:12 (8.33 mg/mL), 1:6 (16.66 mg/mL) & 1:3 (33.33 mg/mL.

Cefepime for injection will be prepared by blending Cefepime hydrochloride

with L-Arginine. Cefepime hydrochloride is acidic and L-Arginine is basic in pH,

while the finished product Cefepime for Injection pH is 4-6.

Method: Equal volume of test sample and LAL reagent is added in a









reagent is taken and following tubes are prepared51






46


directly it inhibits the LAL test and thus shows interference23, 52



standard Endotoxin.


is lower than the expected or enhancement wherein the recovery of

Endotoxin is higher than expected






product.

Significance of product validation is that it gives information on

whether there are any interfering factors in the drug product to the LAL test

and also it gives an idea of the approximate levels of Endotoxin content in

the drug product. It also covers manufacturing of product and formulation of

the product.




change in manufacturing procedures or in vendor.

Phase I: Preliminary Screening / interference Study53



non-interfering dilution (NID) of the product, which is used for the actual

validation (Phase II). The non-interfering dilution (NID) is the first set of PPC that

shows a gel.


47


Cefepime Hydrochloride:

Sample Dilution 1:2 1:4 1:8 1:16 1:32 Unspiked -- -- -- -- --

Spiked -- -- -- -- -- Table: 1

L-Arginine

Sample Dilution 1:1.5 1:3 1:6 1:12 1:24 Unspiked -- -- -- -- --

Spiked -- -- -- -- -- Table: 2

Cefepime Hydrochloride: (Results after adjusting the Acidic pH to the range

of 6-8 with 1 N NaoH).


Spiked ++ ++ ++ ++ ++ Table: 3

L-Arginine (Results after adjusting the Basic pH to the range of 6-8 with 1 N

Hcl)


Spiked ++ ++ ++ ++ ++ Table: 4

Cefepime for Injection:

Sample Dilution 1:3 1:6 1:12 1:24 1:48 Unspiked -- ++ -- -- --

Spiked ++ ++ ++ ++ ++ Table: 5




to the proposed test dilution. Here dilution selected for validation is 1:4. (Hot

spike method).


48

Example of results:

Endotoxin/product Cefepime Hydrochloride

L-Arginine Cefepime for Injection Re

plic

ate

s

0.25

Eu

/mL

0.12

5 Eu

/mL

0.06

25

Eu/m

L 0.

0312

Eu

/mL

0.25

Eu

/mL

0.12

5 Eu

/mL

0.06

25

Eu/m

L 0.

0312

Eu

/mL

0.25

Eu

/mL

0.12

5 Eu

/mL

0.06

25

Eu/m

L 0.

0312

Eu

/mL

1 + + - - + + - - + + - - 2 + + - - + + - - + + - - 3 + + - - + + - - + + - - 4 + + - - + + - - + + - -

Table: 6

Negative product control: --; Geometric Mean = 0.125 EU/ml

Endotoxin/ LRW Replicates 0.125 Eu/mL 0.0625 Eu/mL 0.0312 Eu/mL 0.0156 Eu/mL

1 + + - - 2 + + - - 3 + + - - 4 + + - -

Table: 7

Blank: --; Geometric Mean = 0.125 EU/ml


mean) within +/- one two-fold dilution. Validation is conducted at this dilution

on three batches of product.


49

Figure 1: Cefepime Hcl

Figure 2: Cefepime for Injection

Figure 3: Cefepime for Injection


50

Discussion:

Cefepime Hydrochloride assay shows that there is inhibition up to 1:32 (MVD)

and 1:24 (MVD) in L-Arginine. Due to Inhibition, LAL is unable to detect the

Endotoxins even in spiked sample After analyzing the sample using different

procedures, finally In order to sort out this inhibition problem the acidic pH of

the Cefepime Hydrochloride (1.5-2.7) is adjusted to 6-8 with 1N NaoH and the

basic pH of L-Arginine (10.2-11) is adjusted to 6-8 with 1 N Hcl and both the

samples.

After adjusting the pH Cefepime Hydrochloride assay shows no inhibition up

to 1:2 dilution, 1:1.5 in L-Arginine and the spike recovery at 1:2 and 1:1.5

dilutions onwards. Therefore the NID is 1: 4 (Cefepime hydrochloride), 1:3 (L-

Arginine) and 1:6 (Cefepime for Injection). It is advisable to validate the

product at not less than MVD/4 to take care of any batch to batch variation

during regular production. So MVD/4 dilution is chosen for product validation.

51

3.3 Other Cephalosporin Molecules

Introduction:

During the Research tenure the work was carried out on different molecules

related to Cephalosporin group to understand the factors responsible for

interference during the quantification of Endotoxins in each product. Method

validation was done using different lysate sensitivities and different strengths.


Materials:

Lyophilized Limulus Amoebocyte Lysate of 0.125sensitivity (LAL), Control

Standard Endotoxin 5 Eu/ng (CSE), LAL Reagent Water (LRW) of Endosafe US,

Depyrogenated (250°C for 30 min) 10 X 75 mm assay tubes, 16X100 mm

dilution tubes, pyrogen free Micropipette tips, vortex mixture, 1N NaoH, 1N

Hcl, Ceftriaxone Sodium, Ceftazidime for Injection, Cefazolin Sodium,

Cefepime for Injection, Cefuroxime Sodium, Cefotaxime Sodium, Cephalothin

for Injection, Ceftiofur Injection, Ceftazidime Pentahydrate & Cefepime for

Injection were used for determination of NID by gel clot technique.

The sensitivity of the Lysate (labeled 0.125Eu/mL) was determined by

using known amount of E.coli Control Standard Endotoxin.




units.











52

MVD = (Endotoxin limit X Concentration of sample solution)/ (λ). Where E.L is

the Endotoxin limit of the test sample, which is specified in the individual

monograph in terms of volume or units of active drug (in EU/mg).

3. Ceftriaxone Sodium sample preparation: Batch No: CTN-0209, Potency=25

mg/mL , E.L=0.20 Eu/mg , Lysate sensitivity is 0.125 Eu/mL and MVD = 40.The


1:10 (2.5 mg/mL), 1:5 (5mg/mL) & 1:2.5 (10mg/mL).

4. Ceftazidime for Injection sample preparation: Batch No: CZI-0309,

Potency=25 mg/mL, E.L=0.10 Eu/mg , Lysate sensitivity is 0.125 Eu/mL and MVD

= 20.The following test dilutions are prepared by 1:20 (1.25 mg/mL), 1:10 (2.5

mg/mL), 1:5 (5 mg/mL), 1:2 (12.5 mg/mL) & 1:1 (25 mg/mL).

5. Cefazolin Sodium sample preparation: Batch No: CZS-0309, Potency=25



1:7 (3.57 mg/mL), 1:3 (8.33 mg/mL) & 1:1 (25 mg/mL).

6. Cefepime for Injection (1st) sample preparation: Batch No: CFI-0309,



mg/mL), 1:3 (8.32 mg/mL) & 1:1 (25 mg/mL).

Note: Initial Product pH was 3.5 and then it was adjusted to 7-8 by using 0.1 N

NaoH prepared in LRW.

7. Cefuroxime Sodium sample preparation: Batch No: CXN-0409, Potency=25

mg/mL, E.L=0.10 Eu/mg, Lysate sensitivity is 0.125 Eu/mL and MVD = 20.The


1:5 (5 mg/mL), 1:2 (12.5 mg/mL) & 1:1 (25 mg/mL).

8. Cefotaxime Sodium sample preparation: Batch No: CMN-0409, Potency=25



53

following test dilutions are prepared by 1:10 (2.5 mg/mL), 1:5 (5 mg/mL), 1:2

(12.5 mg/mL) & 1:1 (25 mg/mL).

9. Cephalothin for Injection (1st) sample preparation: Batch No: CFI-0409,



mg/mL), 1:6 (4.16 mg/mL), 1:3 (8.33 mg/mL) & 1:1.5 (16.66 mg/mL).

10. Ceftiofur Injection sample preparation: Batch No: CRI-0509, Potency=30



1:12 (2.5 mg/mL), 1:6 (5 mg/mL) & 1:3 (10 mg/mL).

11. Cephalothin for Injection(2nd) sample preparation: Batch No: CFI-0909,



mg/mL), 1:12 (4.16 mg/mL), 1:6 (8.33 mg/mL) & 1:3 (16.66 mg/mL).

12. Ceftazidime Pentahydrate sample preparation: Batch No: CPH-0109,

Potency=100 mg/mL, E.L=0.10 Eu/mg , Lysate sensitivity is 0.125 Eu/mL and

MVD = 80. The following test dilutions are prepared by 1:80 (1.25 mg/mL), 1:40

(2.5 mg/mL), 1:20 (5 mg/mL), 1:10 (10 mg/mL) & 1:5 (20 mg/mL).

13. Cefepime for Injection(2nd) sample preparation: Batch No: CFI-0208,


= 12. The following test dilutions are prepared by1:12 (2.08 mg/mL), 1:6 (4.16

mg/mL), 1:3 (8.33 mg/mL) & 1:1 (25 mg/mL).

Above mentioned Cephalosporin antibiotics are validated after adjusting

their pH to 6-8, The NID factor was evaluated in the preliminary screening,

variation among them is + two fold.


54

Methods

Equal volume of test sample and LAL reagent is added in a









reagent is taken and following tubes are prepared (USP 32, 2009)






directly it inhibits the LAL test and thus shows interference (van Noordwijk et

al., 1997).



standard Endotoxin.


is below than the expected or enhancement wherein the recovery of

Endotoxin is higher than expected.






product.


55

Significance of product validation is that it gives information on

whether there are any interfering factors in the drug product to the LAL test

and also it gives an idea of the approximate levels of Endotoxin content in

the drug product. It also covers manufacturing of product and formulation of

the product.




change in manufacturing procedures or in vendor.


Phase I: Preliminary Screening / interference Study



non-interfering dilution (NID) of the product. The non-interfering dilution (NID)

is the first set of PPC that shows a gel.

Ceftriaxone Sodium: NID-1:10 (1:20 selected for validation) Sample Dilution 1:2.5 1: 5 1:10 1:20 1:40

Unspiked ++ ++ -- -- -- Spiked ++ ++ ++ ++ ++

Ceftazidime for Injection: NID-1:5 (1:10 selected for validation)


Spiked ++ ++ ++ ++ ++ Cefazolin Sodium: NID-1:7 (1:15 selected for validation)


Spiked ++ ++ ++ ++ ++ Cefepime for Injection(1st): NID-1:1 (1:3 selected for validation)

Sample Dilution 1:1 1:3 1:6 1:12 Unspiked -- -- -- --

Spiked ++ ++ ++ ++ Cefuroxime Sodium: NID-1:5 (1:10 selected for validation)


Spiked ++ ++ ++ ++ ++


56

Cefotaxime Sodium: NID-1:2 (1:5 selected for validation) Sample Dilution 1:1 1:2 1:5 1:10

Unspiked ++ -- -- -- Spiked ++ ++ ++ ++

Cephalothin for Injection(1st): NID-1:6 (1:13 selected for validation)


Spiked ++ ++ ++ ++ ++ Ceftiofur Injection: NID-1:12 (1:24 selected for validation)


Spiked ++ ++ ++ ++ ++ Cephalothin for Injection(2nd): NID-1:26 (1:26 selected for validation)

Sample Dilution 1:3 1:6 1:12 1:26 1:52 Unspiked ++ ++ ++ -- --

Spiked ++ ++ ++ ++ ++ Ceftazidime Pentahydrate: NID-1:20 (1:40 selected for validation)


Spiked ++ ++ ++ ++ ++ Cefepime for Injection(2nd): NID-1:3 (1:6 selected for validation)

Sample Dilution 1:1 1:3 1:6 1:12 Unspiked ++ -- -- --

Spiked ++ ++ ++ ++ Table:1 Results of NID factor in Cephalosporin’s; -- No spike recovery Assay results after preliminary screening with pH adjusting to 6-8 are presented

in Table 1. This assay shows no inhibition from 1:10 dilution onwards in

Ceftriaxone Sodium, 1:5 in Ceftazidime for Injection, 1:7 in Cefazolin Sodium,

1:1 in Cefepime for Injection, 1:5 in Cefuroxime Sodium, 1:2 in Cefotaxime

Sodium, 1:6 in Cephalothin for Injection, 1:12 in Ceftiofur Injection, 1:26 in

Cephalothin for Injection, 1:20 in Ceftazidime Pentahydrate and 1:3 in

Cefepime for Injection and the spike recovery from the NID onwards. It is

advisable to validate the product next to MVD to take care of any batch to

batch variation during regular production in the pharmaceutical industries.


57




to the proposed test dilution. Here dilution selected for validation is refer table

1. (Hot spike method).

Phase: II Results:

Ceftriaxone Sodium Ceftazidime for Injection

Repl

ica

tes

0.

25

Eu/m

L

0.

125

Eu/m

L

0.

0625

Eu

/mL

0.

0312

Eu

/mL

0.

25

Eu/m

L

0.

125

Eu/m

L

0.

0625

Eu

/mL

0.

0312

Eu

/mL

1 + + - - + + - - 2 + + - - + + - - 3 + + - - + + - - 4 + + - - + + - -

Cefazolin Sodium Cefepime for Injection (1st)

Repl

icat

es

0.

25

Eu/m

L

0.

125

Eu/m

L

0.

0625

Eu

/mL

0.

0312

Eu

/mL

0.

25

Eu/m

L

0.

125

Eu/m

L

0.

0625

Eu

/mL

0.

0312

Eu

/mL

1 + + - - + + - - 2 + + - - + + - - 3 + + - - + + - - 4 + + - - + + - -

Cefuroxime Sodium Cefotaxime Sodium

Repl

icat

es

0.

25

Eu/m

L

0.

125

Eu/m

L

0.

0625

Eu

/mL

0.

0312

Eu

/mL

0.

25

Eu/m

L

0.

125

Eu/m

L

0.

0625

Eu

/mL

0.

0312

Eu

/mL

1 + + - - + + - - 2 + + - - + + - - 3 + + - - + + - - 4 + + - - + + - -


58

Cephalothin for Injection (1st) Ceftiofur Injection

Repl

ica

tes

0.

25

Eu/m

L

0.

125

Eu/m

L

0.

0625

Eu

/mL

0.

0312

Eu

/mL

0.

25

Eu/m

L

0.

125

Eu/m

L

0.

0625

Eu

/mL

0.

0312

Eu

/mL

1 + + - - + + - - 2 + + - - + + - - 3 + + - - + + - - 4 + + - - + + - -

Cephalothin for Injection (2nd) Ceftazidime Pentahydrate

Repl

ica

tes

0.

25

Eu/m

L

0.

125

Eu/m

L

0.

0625

Eu

/mL

0.

0312

Eu

/mL

0.

25

Eu/m

L

0.

125

Eu/m

L

0.

0625

Eu

/mL

0.

0312

Eu

/mL

1 + + - - + + - - 2 + + - - + + - - 3 + + - - + + - - 4 + + - - + + - -

Cefepime for Injection (2nd)


Table: 2 Endotoxin/product; Negative product control: --; Geometric Mean = 0.125 EU/ml. Assay results of the products after spiking with known concentration of Endotoxin are presented in Table 2.


Table: 3 Endotoxin/LRW; Negative product control: --; Geometric Mean = 0.125 EU/ml. Assay results of label claim sensitivity of the lysate are presented in Table 3.


59


mean) within +/- one two-fold dilution.

Discussion:

Analytical results of the Cephalosporin group from table 1 after pH

adjustment reveal that NID factor between products tested is between

MVD/2 and MVD/8. In table 2 results shows 100% recovery of the spiked

Endotoxins into the product. It means there is no inhibition or enhancement in

these drug products during quantification of Endotoxins, so there is no

possibility of false positive results and we can apply the same methodology to

understand the behavior of various cephalosporin group drug products

before proceeding for actual testing. It was concluded that NID among the

Cephalosporin antibiotics tested is mostly + two fold.

When ever there is inhibition in assay i.e., the LAL is unable to recover the

spiked Endotoxin also, in that case first Positive product control getting

positive next to inhibitory positive control to be considered as NID. But in case

of non inhibitory assay first negative product control getting negative was

considered as NID.To take care of batch to batch variation, dilution next to

NID to be considered for validation.

Cefepime for Injection & Cephalothin for Injection test samples were

selected from two different manufacturers. In case of Cefepime for Injection

first sample is showing NID at MVD/8 and second sample at MVD/4 and for

Cephalothin for Injection, first sample is showing NID at MVD/4 and second

sample at MVD/2. Even though both manufacturers are manufacturing the

same drug the NID factor is differing do to the changes in the compositions of

the formulations or may be due to raw materials suppliers change. So it is

always advisable to manufacturers to validate their product even a minor

change in the formulation or Raw material vendor change.

Documents

Chapter 3 - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/5681/11/11...Injection (4th generation cephalosporin antibiotic)were used for determination of NID by gel clot technique