sample level ii item-set questions questions 1 - cfa institute

RP-HPLC as an

analytical method for

the determination of

bendamustine and its

related impurities in

bulk drug

6.1 Introduction

Bendamustine hydrochloride (BMH) chemically known as 4-{5-[bis-(2-chloroethyl) amino]-

1-methyl- 1Hbenzimidazol-2-yl} butanoic acid, is an active nitrogen mustard [1]. The IUPAC

names of its impurities were impurity-A 4-6-[2-chloro ethyl)-2-hydroxy ethyl amino)3 methyl-

benzimidazolyl-2)-butyric acid (HP-1), impurity-B 4-(-5-[(4-5-(bis(2-hydroxyethyl)amino)-1-

methyl-1H-benzo[d]imidazol-2-yl)butanoic acid and impurity-C isopropyl 4-(5-(bis(2-

chloroethyl)amino-1-methyl-1-H-benzol[d]imidazol-2yl)butanoate. It was used for the treatment

of patients with chronic lymphocytic leukemia [2]. It contains mechlorethamine group and

benzimidazole heterocyclic ring with a butyric acid substituent. Mechlorethamine and its

derivatives form electrophilic alkyl groups. These groups form covalent bonds with electron-rich

nucleophilic moieties, resulting in interstrand DNA cross links. The bi functional covalent

linkage can lead to cell death via several pathways [3]. It was active against both quiescent and

dividing cells [4-7]. It was approved by the US food and drug administration (FDA) for chronic

lymphocytic leukaemia in march 2008 [8, 9].The chemical structure of bendamustine shown in

the Figure-6.1.

Figure-6.1: Bendamustine hydrochloride

Its empirical molecular formula was C16H21Cl2N3O2•HC1 and the molecular weight 394.7.

It was first synthesized in 1963 by Ozegowski and Krebs in East Germany (the former German

Democratic Republic). Until 1990 it was available only in East Germany. The German

investigators found that it was useful for treating chronic lymphocytic leukemia, Hodgkin’s

disease, non-Hodgkin’s lymphoma, multiple myeloma and lung cancer.

Bendamustine was marketed under the trade name Ribomustin by Astellas Pharma. It was

used alone or in combination with other anti cancer agents for indolent non-hodgkin's

lymphoma, multiple myeloma, and chronic lymphocytic leukemia. SymBio Pharmaceuticals Ltd

holds exclusive rights to develop and market bendamustine in Japan and in selected Asia Pacific

countries.

In March 2008,Cephalon received approval from the United States Food and Drug

Administration to market bendamustine in the US, where it sold under the trade name treanda for

the treatment of chronic lymphocytic leukemia[10].

In October 2008, the FDA granted further approval to market treanda for the treatment of

indolent B-cell non-Hodgkin's lymphoma that has progressed during or within six months of

treatment with rituximab or a rituximab-containing regimen [11]. The pH of the reconstituted

solution was 2.5 - 3.5. Each 25-mg vial contains 25 mg of bendamustine hydrochloride and 42.5

mg of mannitol. Each 100-mg vial contains 100 mg of bendamustine hydrochloride and 170 mg

of mannitol. It was supplied as a sterile non-pyrogenic white to off-white lyophilized powder in a

single use vial.

Figure-6.2: Bendamustine hydrochloride vial 100mg

Bendamustine is water soluble microcrystalline powder with amphoteric properties. It acts

as an alkylating agent causing intra-strand and inter-strand cross-links between DNA bases.

After intravenous infusion, it is extensively metabolised in the liver by cytochrome p450. More

than 95% of the drug is bound to protein. Only free bendamustine is active. Its elimination is

biphasic with a half-life of 6–10 minutes and a terminal half-life of approximately 30 minutes. It

is eliminated primarily through the kidneys.

Figure-6.3: Clinical mechanism of bendamustine hydrochloride with DNA

In the available literature, many analytical procedures have been reported for the

quantitative determination of bendamustine in pure form as well as in pharmaceutical dosage

formulation by different analytical techniques like high performance liquid chromatography [13-

16], and spectrophotometric methods [17-18]. Annapurna et.al proposed a stability-indicating

liquid chromatographic method for the determination of bendamustine hydrochloride in

parenterals. Reversed phase chromatography was performed using C18 (250 mm × 4.6 mm, 5 µm

particle size) column with acetonitrile: tetra butyl ammonium hydrogen sulphate (80:20, V/V) as

mobile phase at a flow rate of 0.8 mL/min. with UV detection at 233nm [14]. Pavani priya et.al

developed stability indicating RP-HPLC method for the determination of bendamustine in

parenterals by using Agilent Zorbax poroshell C18 RP column. The mobile phase of water and

acetonitrile (with 0.01% TFA), mixed in the ratio of 50:50 (pH 6.9-7.2) was used in this study

[15]. A simple, rapid and sensitive spectrophotometric method was developed for the

determination of bendamustine hydrochloride in phosphate buffer (pH 9.0) by Mathrusri

Annapurna et.al. [18]. Some HPLC methods were also reported in literature for the determination

of bendamustine [19-31].

Though large numbers of assay methods are available in literature for bendamustine, only

very few of them are standard, sensitive and selective. In view of the importance of

bendamustine in drug formulation in the treatment of chronic lymphocytic leukemia, a more

simple, sensitive, selective and robust method is needed for its validation in bulk drug

formulations. All the reported methods were used for the determination of bendamustine in bulk

and formulations but no method was reported for the determination of bendamustine and its

related impurities. We are now reporting a simple sensitive and selective RP-HPLC method for

the validation of bendamustine and its related impurities which is robust and rugged method.

6.2 Experimental:

6.2.1 Chemicals, reagents and samples:

The standard, samples of bendamustine and known related impurities such as impurity-A,

impurity-B and impurity–C were received from Bio-Leo Labs India (P) Ltd., Hyderabad. HPLC

grade methanol, acetonitrile and trifluroacetic acid were purchased from Merck, Mumbai, India.

High purity water was prepared by using Millipore Milli-Q plus water purification system. The

purity of all samples and impurities used in this study was greater than 99%.

6.2.2 Instrumentation

For initial method development studies Waters prominence HPLC system was employed.

This was equipped with a quaternary UFLC LC-20AD pump, DGU-20A5 degasser, SPD-M20A

diode array detector,SIL-20AC auto sampler,CTO-20AC column oven and CBM-20A

communications bus module. Agilent 1200 series with high pressure liquid chromatographic

instrument provided with Auto sampler and VWD UV detector with thermostatted column

compartment connected with EZ Chrom software was employed for the validation of the drug

and its related impurities. The analysis was carried out on Zorbax SB-C18, 4.6 mm x 25 cm

column with 5µm particle size.

6.2.3 Standard and sample solutions:

6.2.3.1. Preparation of standard solution:

20.0 mg of bendamustine hydrochloride standard were accurately weighed and transferred

into a 20 ml Volumetric flask dissolved and diluted to the Volume with methanol. 2.0 ml of this

solution was further diluted to 100 ml with methanol. The resultant solution was filtered through

0.45 micron filter.

6.2.3.2 Sample preparation:

25.0 mg of bendamustine hydrochloride sample were weighed into a 25 ml Volumetric

flask dissolved in methanol and diluted to the Volume with methanol. The resultant solution was

filtered through 0.45 micron filter.

6.3 Evaluation of system suitability:

The system suitability was evaluated by injecting a known Volume of sample containing a

known amount of bendamustine into chromatograph and calculated the number of theoretical

plates and asymmetry of the chromatogram. The number of theoretical plates was found to be

greater than 3000 and the tailing factor of bendamustine was calculated and found to be more

than 2.0 which showed that the selected column is suitable for the analysis.

6.4 Results and discussion:

6.4.1 Method development and optimization:

The method development was initiated with the solubility study of bendamustine. Based on

the solubility studies, methanol was chosen as solvent for the preparation of sample solutions.

Bendamustine is polar in nature due to the presence of one –COOH group. Hence, non polar

silica based C18 was selected for developing reverse phase high performance liquid

chromatogram. From the molecular structure, it was observed that, there is chromophore group

present in bendamustine. Hence there is possibility for its UV–Visible detection. The UV

experiment was performed on bendamustine, which showed maximum absorbance at 230nm.

To arrive at the optimal chromatographic conditions suitable for the validation of

bendamustine and its related impurities, various trail chromatograms were recorded with

different conditions.

Trail-1

The first trail method was performed by using isocratic mode and mobile phase 0.1%

trifluroacetic acid in water and acetonitrile in the ratio 90:10v/v as the mobile phase. The

injection Volume was 10µL.

Column : Zorbax SB-C18,(4.6 mm x 25 cm) 5µm particle size

Pump mode : Isocratic

Flow rate : 1.0 ml/min

Detection wavelength : UV , 230 nm

Injection Volume : 10µL

Column temperature : 300C

Run time : 60 min

Mobile phase : 0.1% Trifluroacetic acid in water: acetonitrile (90:10v/v)

Diluent : Methanol

In this trail there was no elution of impurity peaks up to a run time of 60 minutes and asymmetry

of bendamustine peak was more than 2.0

Trail-2

To over the limitations of the above trail method, the experiment was repeated by changing

the mode of mobile phase, composition and isocratic mode to gradient mode, keeping the

remaining conditions constant. The mobile at present is a gradient mixture of mobile phase-A

(80:20v/v) and mobile phase-B (60:40v/v).


Pump mode : Gradient





Run time : 60 min

Mobile phase A : 0.1% Trifluroacetic acid in Water: Acetonitrile (80:20v/v)

Mobile phase B : 0.1% Trifluroacetic acid in Water: Acetonitrile (60:40v/v)

Diluent : Methanol

In this trail there was elution of bendamustine and its known impurities but peak shapes of

bendamustine and its impurities were not symmetrical.

Trail -3

To achieve the peak symmetries for bendamustine and its impurities, both the mobile phase

compositions were changed as follows and the experiment was repeated under the optimal

conditions.


Pump mode : Gradient





Run time : 60 min

Mobile phase A : 0.1% Trifluroacetic acid in Water: Acetonitrile (90:10v/v)

Mobile phase B : 0.1% Trifluroacetic acid in Water: Acetonitrile (50:50v/v)

Diluent : Methanol

In this trail bendamustine peak was eluted at 24 minutes and the impurities-A, B and C of

bendamustine were well separated with main bendamustine peak with retention times of 15, 30

and 38 minutes and all the peaks were obtained with excellent symmetry.

Finally, satisfactory separation with better peak shape was achieved by employing the

optimal conditions of trail-III within a reasonable retention time with gradient mode with a flow

rate of 1.0mL/min at temperature 300C.

6.5 Method validation:

In order to determine bendamustine and its related impurities, the method was validated as

per the ICH guidelines [32-33] individually in terms of system suitability, specificity, precision,

accuracy, linearity, robustness, limit of detection and limit of quantification (LOD and LOQ) and

solution stability.

6.5.1 System suitability test:

The system suitability was studied by injecting diluted blank one injection and diluted

standard six replicate injections. The RSD from six replicate injections of diluted standard

preparation was calculated. The system suitability results are given in Table-6.1

Table-6.1: Results of system suitability

System suitability parameters

Result

Acceptance criteria as per USP

Theoretical plates 778991 > 3000

Tailing factor 1.1 < 2.0

% RSD for six injections 0.9 < 5.0%

.

6.5.2 Specificity of method with related substances:

Specificity is the ability of the method to accurately measure the analyte response in the

presence of all potential sample components. The response of the analyte in test mixtures

containing the analyte and all potential components is compared with the response of a solution

containing only analyte. For specificity determination, solution containing diluent and all related

substances of bendamustine was prepared by mixing in suitable proportions. Then diluent,

standard preparation, sample preparation, sample spiked with impurities were injected into the

chromatograph and the peak homogeneity was verified for bendamustine and its related

substances using EZ Chrom software. The specificity experiment results are given in the Table-

6.2. The specificity chromatograms are shown in Figures-6.4

Table-6.2: Results of specificity experiment

S.No. Name of the impurity/analyte Peak purity Retention time

1 Bendamustine 1.00000 24.109

2 Impurity-A 1.00000 15.306

3 Impurity-B 1.00000 30.505

4 Impurity-C 1.00000 38.638

6.5.3 Precision:

6.5.3.1 System precision (Repeatability):

Six replicate aliquots of sample solution of bendamustine, spiked with impurities, were

injected into RP-HPLC system and the chromatograms were recorded for checking the

performance of the system under the chromatographic conditions on the day tested and

calculated the relative standard deviation. The RSD value obtained was 0.8%, which showed the

good precision of the system. The system precision results are shown in Table-6.3

Table-6.3: System precision of bendamustine hydrochloride

Injection No.

Bendamustine hydrochloride

Retention time Area response

1 24.833 88860

2 24.840 88250

3 24.841 87923

4 24.828 86902

5 24.824 87794

6 24.825 87359

Mean 24.832 87848

% RSD 0.0 0.8

6.5.3.2 Intermediate precision (Ruggedness):

The intermediate precision also called as ruggedness, is the inter-day variation. It is defined

as the degree of reproducibility obtained by following the same procedure as mentioned for

method precision experiment. The ruggedness of the test method was demonstrated by carrying

out precision study in six preparations of sample, a single batch sample by different analysts,

different columns on different days using different instruments and calculated the percentage of

impurities. The RSD for impurities from six spiked sample preparations were found to be 0.7 for

impurity-A, 0.8 for impurity-B and 0.8 for impurity-C showing high ruggedness of the proposed

method. The validated intermediate precision results are given in Table-6.4

Table-6.4: Results of intermediate precision

Injections Impurity- A Impurity -B Impurity -C

1 0.217 0.182 0.175

2 0.217 0.185 0.172

3 0.218 0.186 0.175

4 0.214 0.186 0.175

5 0.217 0.185 0.175

6 0.215 0.184 0.173

Mean 0.216 0.185 0.174

% RSD 0.7 0.8 0.8

6.5.4 Accuracy:

The accuracy of the proposed method was tested by preparing sample solutions with known

quantities of impurities-A, B and C at the level of LOQ, 50%, 100%, 150%, 200% and 300% of

target concentration (i.e., 1.0 % of test concentration).The chromatograms were recorded and the

recovery percentages were evaluated from the peak areas. The results are shown in Table-6.5

Table-6.5: Accuracy results of bendamustine hydrochloride and its impurities

Levels

Mean recovery (%)

Bendamustine Impurity-A Impurity-B Impurity-C RSD (%)

LOQ 101.5 99.7 101.0 99.9 0.9

50% 100.2 101.0 99.7 99.2 0.8

100% 101.5 99.9 101.0 99.7 0.9

150% 99.9 99.7 99.2 99.1 0.4

200% 101.5 99.2 98.8 99.2 1.5

300% 99.8 99.7 102.1 100.5 1.1

6.5.5 Linearity:

The concentration ranges in which bendamustine, its impurity-A, impurity-B and impurity-

C can be determined with good accuracy were evaluated by preparing calibration plots between

the concentration of the analyte and peak areas. Six different aliquots of standard solutions were

injected into the RP-HPLC chromatograph and the chromatograms were recorded. Peak areas of

the resultant chromatogram were plotted against the concentration of the analyte. The

experimental data obtained are shown in Table-6.6 and the resultant linear plots obtained for

these data are given in Figure-6.5.

Table 6.6: Results of linearity experiment of bendamustine and its related impurities

a) Bendamustine b) Impurity-A

Level Amount of

bendamustine(ppm)

Area

Response

1 0.0129 403

2 0.1932 7321

3 0.4895 18611

4 0.7729 30367

5 1.1723 48577

6 1.4814 61200

Correlation coefficient 0.9995

C) Impurity-B D) Impurity-C

The data was subjected to statistical analysis and the results of these analyses are presented in

table 6.6. The values of different statistical analyses like correlation coefficient intercept,

residual sum square and relative standard deviation confirm high precision and accuracy of the

proposed method.

6.5.6 Limit of detection and limit of quantification:

Level Amount of

impurity-A(ppm)

Area

Response

1 0.0195 616

2 0.1461 4482

3 0.2923 9140

4 0.7404 23477

5 1.169 38146

6 1.4807 48508


Level Amount of

impurity-C(ppm) Area Response

1 0.0193 1097

2 0.1451 5874

3 0.2902 11670

4 0.7351 27992

5 1.1607 44194

6 1.4702 55895


Level Amount of

impurity-B(ppm)

Area

Response

1 0.0196 858

2 0.1467 5410

3 0.2934 11388

4 0.7434 29078

5 1.1737 46234

6 1.4867 59331


Limit of quantification and limit of detection were established for bendamustine and its

related impurities based on slope ratio method. The detection of an individual analytical

procedure is the lowest amount of analyte in a sample, which can be detected not necessarily

quantitated as an exact value. The quantification limit of an individual analytical procedure was

the lowest amount of analyte in a sample which can be quantitatively determined with suitable

precision and accuracy. The LOD & LOQ results of bendamustine and its impurities were given

in the Table-6.7.

Table 6.7: LOQ and LOD results of bendamustine and its related impurities

S.No.

Analyte/impurity

LOD concentrations

(ppm)

LOQ concentrations

(ppm)

1. Bendamustine 0.092 0.306

2. Impurity-A 0.072 0.239

3. Impurity-B 0.127 0.383

4. Impurity-C 0.127 0.226

6.6 CONCLUSION

The liquid chromatographic method with gradient elution developed for determination of

bendamustine hydrochloride and its impurities A, B and C in the bulk drug, was fully validated

and proved to be reliable, sensitive, accurate and precise .The method has higher sensitivity

towards the determination of impurities and it is the first time that such method appears in the

literature and can be useful for routine analysis and quality control of bendamustine

hydrochloride in the relevant forms. The method can be used for quality assurance of

bendamustine hydrochloride in bulk drugs and can be extended to validate the pharmaceutical

formulations.

References:

[1] Neil M.J.O. The Merck Index, Merck Research Laboratories, Whitehouse Station, 2006.

[2] Friedberg J. W., Cohen P., Chen L., et al., Bendamustine in Patients with Rituximab-

Refractory Indolent and Transformed Non-Hodgkin's Lymphoma: Results from a Phase II

Multicenter, Single-Agent Study, Journal of Clinical Oncology, 26(2), 2008, 204-210.

[3] Lissitchkov T., Arnaudov G., Peytchev D., Merkle Kh., Phase-I/II Study to Evaluate Dose

Limiting Toxicity, Maximum Tolerated Dose, and Tolerability of Bendamustine HCl in Pre-

Treated Patients with B-Chronic Lymphocytic Leukaemia (Binet Stages B and C) Requiring

Therapy, Journal of Cancer Research and Clinical Oncology, 132(2), 2006, 99-104.

[4] Teichert J., Sohr R., Baumann F., Hennig L., et al., synthesis and characterization of some

new phase-ii metabolites of the alkylator bendamustine and their identification in human bile,

urine, and plasma from patients with cholangiocarcinoma, Drug Metabolism and Disposition,

33(7), 2005, 984-992.

[5] Matt Kalaycio, Clinical Experience with Bendamustine: A new treatment for patients with

chronic lymphocytic leukemia, Clinical Leukemia, 2(4), 2008, 223-229.

[6] Rasschaert M., Schrijvers D., Van den Brande J. et al., A Phase I Study of Bendamustine

Hydrochloride Administered Once Every Three Weeks in Patients With Solid Tumors,

British Journal of Cancer, 96, 2007,1692-1698.

[7] O'Brien S, Moore JO, Boyd TE, et al. 5-year survival in patients with relapsed or refractory

chronic lymphocytic leukemia in a randomized, phase III trial of fludarabine plus

cyclophosphamide with or without oblimersen, Journal of Clinical Oncology, 27(31), 2009,

5208-5212.

[8] Rassenti LZ, Huynh L, Toy TL, et al. ZAP-70 compared with immunoglobulin heavy-chain

gene mutation status as a predictor of disease progression in chronic lymphocytic leukemia.

New England journal of medicine, 351(9), 2004, 893-901.

[9] http://www.cancer.gov/cancertopics/druginfo/fdabendamustine-hydrochloride-indications

[10] http://www.chemblink.com

[11] Schroers R, Griesinger F, Trümper L, et al. Combined analysis of ZAP-70 and CD38

expression as a predictor of disease progression in B-cell chronic lymphocytic leukemia,

European Journal of Haematology, 19(5), 2005, 750-758.

[12] Wierda WG, O'Brien S. Chronic lymphocytic leukemias, Lawrence TS, Rosenberg SA,

DeVita, Hellman, and Rosenberg's Cancer, Philadelphia, Pa: Lippincott Williams &

Wilkins, Principles and Practice of Oncology, 9th edition, 2011, 1973–1987.

[13] Ivanka Pencheva, Anita Bogomilova, Neli Koseva, Danka Obreshkova, Kolio Troev, HPLC

Study on the Stability of Bendamustine Hydrochloride Immobilized onto

Polyphosphoesters, Journal of Pharmaceutical and Biomedical Analysis, 48(4), 2008,

1143-1150.

[14] Mathrusri Annapurna M., Venkatesh B., Anusha S. and Neelima B. Stability-Indicating

Liquid Chromatographic Method for the Determination of Bendamustine Hydrochloride in

Parenterals, Research Journal of Chemical Sciences, 2(9), 2012, 72-78.

[15] Pavani Priya B, Vasanth PM, Ramesh T, Ramesh Malothu, a stability indicating rp-hplc

method for the determination of bendamustine hcl in parenterals, International Journal of

Bioassays, 2 (1), 2013, 260-264.

[16] Prasanna, Puranik, Preparation and evaluation of bendamustine hydrochloride non aqueous

formulations, International journal for pharmaceutical research scholars, 2(1), 2013,

1110-1133.

[17] Mathrusri Annapurna M., Pavani S., Anusha S., Harika Mahanti and Venkatesh B, New

analytical methods for the determination of bendamustine hydrochloride – an anti-

neoplastic drug, Journal of Chemical and Pharmaceutical Research,4(3), 2012,1696-1701.

[18] Mathrusri Annapurna, Pavani, Anusha, and Harika Mahanti, Derivative

spectrophotometric methods for the determination of bendamustine hydrochloride,

journal of applied pharmaceutical science, 2 (11), 2012, 139-142.

[19] Wierda WG, Lamanna N, Weiss MA. Chronic lymphocytic leukemia. In: Pazdur R,

Wagman LD, Camphausen KA, Hoskins WJ, eds. Cancer Management: A Multidisciplinary

Approach, 11th ed. Lawrence, KS: CMPMedica, 2008, 843–858.

[20] Grever MR, Andritsos LA, Lozanski G. Chronic lymphoid leukemias, Abeloff's Clinical

Oncology, 4th ed. Philadelphia, Pa: Elsevier, 2008, 2293–2308.

[21] National Comprehensive Cancer Network (NCCN), Clinical Practice Guidelines in

Oncology, Non-Hodgkin's Lymphomas, 2012.

[22] American Cancer Society. Cancer Facts and Figures. Atlanta, Ga:American Cancer Society,

2013.

[23] "Cephalon press release - Cephalon Receives FDA Approval for TREANDA, a novel

chemotherapy for chronic lymphocytic leukemia", 2008.

[24] T Lissitchkov; G Arnaudov; D Peytchev, Merkle Kh, Phase-I/II study to evaluate dose

limiting toxicity, maximum tolerated dose, and tolerability of bendamustine HCl in pre-

treated patients with B-chronic lymphocytic leukaemia (Binet stages B and C) requiring

therapy, Journal of Cancer Research and Clinical Oncology, 132(2), 2006, 99.

[25] Ivanka Pencheva; Anita Bogomilova; Neli Koseva; Danka Obreshkova; Kolio Troev, HPLC

study on the stability of bendamustine hydrochloride immobilized onto polyphosphoesters,

Journal of Pharmaceutical and Biomedical Analysis,48,2008,1143-1150.

[26] JW Friedberg; P Cohen; L Chen; KS Robinson; A Forero-Torres; AS La Casce; LE Fayad;

A Bessudo; ES Camacho; ME Williams; RH van der Jagt; JW Oliver; BD Cheson,

Bendamustine in Patients With Rituximab-Refractory Indolent and Transformed Non-

Hodgkin's Lymphoma: Results From a Phase II Multicenter, Single-Agent Study, Journal

of Clinical Oncology, 26(2), 2008, 204-210.

[27] J Teichert, R Sohr, F Baumann, L Hennig, K Merkle, K Caca, R Preiss, Synthesis and

characterization of some new phase II metabolites of the alkylator bendamustine and their

identification in human bile, urine, and plasma from patients with cholangiocarcinoma,

Drug Metab Dispos, 33, 2005, 984-992.

[28] J Teichert; F Baumann; Q Chao; C Franklin; B Bailey; L Hennig; K Caca; K Schoppmeyer;

U Patzak; R Preiss, Characterization of two phase I metabolites of bendamustine in human

liver microsomes and in cancer patients treated with bendamustine hydrochloride,

Cancer Chemotherapy Pharmacology, 59,2007, 759-770.

[29] M Rasschaert; D Schrijvers; J Van den Brande; J Dyck; J Bosmans; K Merkle. J ermorken,

A phase I study of bendamustine hydrochloride administered day 1+2 every 3 weeks in

patients with solid tumours , British Journal of Cancer, 96,2007;1692- 1698.

[30] Preiss R, Sohr R, Matthias M, Brockmann B, Huller H. The pharmacokinetics of

bendamustine (Cytostasane) in humans. Die Pharmazie, 40(11), 1985, 782–784.

[31] He L, Grecula JC, Ling Y, Enzerra MD, Ammirati M, Kendra K, Cavaliere R, Mayr N,

McGregor J, Olencki T, Mrozek E, Matharbootham M, Oluigbo C, Phelps MA,

Development and validation of sensitive liquid chromatography/tandem mass spectrometry

method for quantification of Bendamustine in mouse brain tissue, Journal of

chromatography-B, Analytical technologies in the biomedical and life sciences, 905, 2012,

141-144.

[32] Validation of Analytical Procedures: Methodology (Q2B), ICH Harmonized Tripartite

Guidelines, Geneva, 1996.

[33] International Conference on Harmonization of Technical Requirements for the Registration

of Pharmaceutical for Human Use: Validation of Analytical procedures, Text and

methodology, 2005.

sample level ii item-set questions questions 1 - cfa institute

Documents