Trouble Shooting During Bio Analytical Estimation of Drug and Metabolites Using LC-MSMS a Review[1]

8/2/2019 Trouble Shooting During Bio Analytical Estimation of Drug and Metabolites Using LC-MSMS a Review[1]

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Journal of Advanced Pharmaceutical Technology & Research Vol. 1 (1), Jan-Mar, 2010 ISSN 0976-2094

Available online on www.japtr.org 1

TROUBLE SHOOTING DURING BIOANALYTICAL ESTIMATION OF DRUG

AND METABOLITES USING LC-MS/MS: A REVIEW

Ganesh N. Sharma*1, Man Mohan Singhal

1,K.K.Sharma

2, Jyotsana Sanadya

3

1. School of Pharmaceutical Sciences, Jaipur NationalUniversity, Jaipur, Rajasthan, (India)2. LBS College of Pharmacy, Tilak Nagar, Jaipur, Rajasthan, (India)3. Maharishi Arvind Institute of Pharmacy, Mansarovar, Jaipur, Rajasthan, (India)

Corresponding Authors E-mail: - [email protected]

Received: 08th January 2010 Revised: 11th February 2010 Accepted: 01st March 2010

AbstractBioanalysis frequently involves the measurement of very low analyte

concentrations in complex and potentially variable matrices. An initial attempt has been

made to apply a risk management tool to the bioanalytical method development like

selection of spiked plasma volume, selection of internal standard to minimize processing

error, selection of medium and extraction procedure, setting of mobile phase and pH,

determination of chromatographic conditions etc. and to minimize instrumental error

like; ion suppression and matrix effect.

Key Words:Bioanalysis, LC-MS/MS, Ion suppression, Matrix effect.

Introduction

The pharmacological response is

generally related to the concentration of drug

at receptor site [1] and the availability of a

drug from a dosage form is a critical element

of drug efficacy [2]. However; drug

concentrations usually, cannot be readily

measured directly at the site of action,

therefore most bioavailability (BA) studies

involve the determination of drug

concentration in the biological matrix [3]

like blood, plasma, urine etc. This is based

on the premise that the drug at site of action

is in equilibrium with drug in blood. It is

therefore possible to obtain an indirect

measure of drug response by monitoring

drug level in biological matrix.

The bio-analysis is employed for the

quantitative determination of drug &

metabolites in biological fluids play a


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significant role in evaluation of

bioequivalence (BE),

pharmacokinetic (PK)

and toxicological studies. It is the way of

comparative studies between two or more

formulations of the similar drug products.

One of the recent important instrumental

developments, giving high selectivity and

sensitivity for complex samples, is the

hyphenation of liquid chromatography

(HPLC) with mass spectrometry (MS) [4] i.e.

LC-MS or MS is combination of high

performance liquid chromatography (HPLC).

Chromatography is a non-destructive

procedure for resolving a multi component

mixture of trace, minor or major constituent

into its individual fractions. This may be

applied both quantitatively and qualitatively.

LC-MS/MS is the best method, when the

detector has to be more sensitive, more

specific or unknowns have to be identified [5].

An advantage is that, at least for some of the

LC-MS / MS techniques, the development of a

method needs no compromise for adaptation

to MS; drawbacks are such as the added

degree of complexity in instrumentation and,

in studies requiring precise quantitative work,

the need for internal standards [6].

Bioanalysis

Bioanalysis is employed for the

quantitative determination of drug &

metabolites in biological fluids play a

significant role in evaluation of BE,

Pharmacokinetic (PK) and toxicological

studies. It is the way of comparative studies

between two or more formulations of the

similar drug products.

Applications of bioanalysis

1. To identify and /or quantify illicit drugsin biological samples for the detection of

substances of abuse

2. To assay foreign substances in biologicalmaterials, sometime postmortem for

evidence of poison in toxicological and

forensic use.

3. To analyze trace elements and toxicsubstances in natural science and

environmental research.

4. To monitor the concentration of drugsand metabolites in tissues, blood,

plasma, serum and urine specimens for abetter understanding of the

pharmacology and toxicity of drugs.

5. To apply therapeutic drug monitoring inthe management of the drug treatment in

patients.

LC-MS/MS

Liquid chromatography and mass

spectrometry is two segment of a LC-

MS/MS system. The sample under

investigation has to be introduced into the

ionisation source of the instrument. Inside

the ionisation source, sample molecules are


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ionised. These ions are extracted into the

analyser region of the mass spectrometer

where they are separated according to their

mass (m) to charge (z) ratios (m/z). The

separated ions are detected and this signal sent

to a data system where the m/z ratios are

stored together with their relative abundance

for presentation in the format of an m/z

spectrum.

CHROMATOGRAPHY

Chromatography is a non-destructive

procedure for resolving a multi component

mixture of trace, minor or major constituent

into its individual fractions. This may be

applied both quantitatively and qualitatively.

High performance liquid

chromatography

HPLC is an analytical technique for

the separation and determination of organic

and inorganic solutes in any sample,

especially biological, pharmaceutical, food,

environmental, and industrial. [7] HPLC is

more versatile since it is not limited to volatile

and thermally stable samples, and the choice

of mobile phase and stationary phase is

wider.[8]Mass Spectrometry

Mass spectrometers are an analytical

tool used for measuring the molecular weight

(MW) of a sample. The principal of MS is the

production of ions from analysed compounds

that are separated or filtered on the basis of

their mass to charge ratio (m / z). [9]

Structural information can be generated

using certain types of mass spectrometers,

usually tandem mass spectrometers, and this

is achieved by fragmenting the sample and

analysing the products generated.

Figure.1: Components of an LC system.

Tandem (MS-MS) mass

spectrometers are instruments that have

more than one analyser and so can be used

for structural and sequencing studies. Two,

three and four analysers have all been

incorporated into commercially availabletandem instruments, and the analysers do

not necessarily have to be of the same type,

in which case the instrument is a hybrid one.

More popular tandem mass spectrometers

include those of the quadrupole-quadrupole,


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magnetic sector-quadrupole, and more

recently, the quadruple-time-of-flight

geometries. [10]

Figure.2 Typical flow diagram of a Mass

Spectrometer

Figure.3 Typical LC-MS/MS configuration

Critical points in bioanalysis with

LC-MS/MS

In order to determine the accurate

concentration of drug and metabolites inbiological matrix there are so many

challenges. These challenges make the

overall process difficult. The major

challenges are as:

What would be the exactchromatographic conditions including

selection of column, mobile phase, flow

conditions, sample preparation method

and detection system?

Is the developed method precise,accurate and reproducible? Is it robust

with regard to column, machine and

analyst? To solve these problems, we

have to validate [11] the developed

method with regard to both the parent

drug and metabolite.

Development of analytical method

Development of a suitable method

for analysis of drug is the most important

step in analytical studies, since the behavior

of the drugs in biological matrixes depends

on the levels of interferences that interactwith the active molecule. Another aspect to

be highlighted is the change of the

components of the biological matrix subject

to storage, process, taking into consideration

time and temperature. Thus degradation


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products, complexing, oxidation, metabolites

and other substances change the response of a

method if this is not sufficiently selective for

the studies with fresh and aged biological

matrixes.

An idea regarding the method to be

used for analysis, we can obtain from the

various available research papers, literature

survey etc. Literature surveys should be made

with regard to following in mind:

I. Maximum plasma concentration (Cmax) ofthe drug and metabolite: knowing Cmax

helps the establishment of target

sensitivity of method.

II. Physiochemical properties of the drug andmetabolite: following properties aid to

guess and select proper extraction and

sample preparation procedure.

a) Molecular weight (MW)b) Solubilityc) Structured) Dissociation constant (pKa)e) Melting point.

Determination of lower and upper

limit of quantification

The lowest concentration of an analyte

or lower limit of quantification (LLOQ) in a

sample that can be quantitatively determined

with an acceptable precision and accuracy is

usually 1/20th of the Cmax value. After

calculating the ULOQ and LLOQ values [12]

we prepare standard workings stock solution

(main stock) of highest and lowest

concentration. Now from these main stocks,

we prepare different solutions of

intermediate concentration.

Setting of mobile phase

A successful chromatographic

separation depends upon differences in the

interaction of the solutes with the mobile

phase and the stationary phase, and in liquid

chromatography, choice and variation of the

mobile phase is of critical importance in

achieving optimum efficiency.

Establishment of extraction

procedure

I. Physiochemical property of drugsII. Selection of solvent for extraction: A

good solvent contains following

characteristics:

Drug should be soluble properly in thesolvent.

It should not be flammable andhazardous.

It should be easily available andshould not be of high cost.

It should be easily evaporated, so thatdrug can be re-obtained from the

solution.

III. Selection of medium (acidic/ basic orneutral)


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IV. Selection of extraction procedure i.e. eitherliquid-liquid extraction (LLE), protein

precipitation (PP), or solid phase extraction

(SPE).

Selection of plasma volume spiked

Minimum plasma volume which provided

sufficient recovery of both drug and the

metabolite is used in plasma blank sample.

Recovery of drug and metabolites are

calculated by the formula;

Mean peak response

(Extracted sample)

% recovery = _______________ x 100

Mean peak response

(Un-extracted sample)

Selection of drug and metabolite

volume to spike

The volume of drug and metabolite to

be added depend upon the volume of plasma

spiked. Drug and metabolite concentration is

normally 5 % of the spiked plasma volume.

For example, if the spiked plasma volume is

500 l, so the volume of drug and metabolite

to be added will be 25l of each.

Selection of internal standard

To determine whether the sample

prepared is with least error, internal standard

is added in plasma. Internal standard is the,

compound of known purity, which, it does not

interfere in the analysis. Internal standard

should be chemically and physically similar

with the drug, as well as it should not have

any chemical reaction with drug and should

be of high purity. Generally, internal

standard is selected, from the same category

of the drug. For example; In the case of

Losartan and Losartan Carboxylic Acid

bioanalytical study, valsartan may be used

as the internal standard.

Optimization of matrix effect

The US-FDA guidance for industry

on bioanalytical method validation requires

the assessment of matrix effect during

method validation for quantitative

bioanalytical LC-MS/MS methods. A matrix

effect [13] is defined as the analyte

ionization suppression or enhancement at

the presence of the matrix components thatcould originated from the endogenous

compounds, metabolites and co-

administered drugs. More recently matrix

effects from internally standard dosing

vehicle and mobile phase additives and

plastic tubes also reported.

This step is usually necessary to

remove matrix components such as proteins,

salts and other organic compounds in

biological matrices which otherwise may

impose interferences or ionization

suppressions to the analytes.


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Ion suppression

Ion suppression in liquid

chromatography coupled with mass

spectrometry (LC-MS/MS) can occur, when a

co-eluted compound suppresses the ionization

of the sample molecules in a mass

spectrometers source. This ion suppression is

analogous to the large garbage peak for

unretained material common at the beginning

of chromatograms monitored by UV

detection. Ion suppression, like other

chromatographic interferences, compromises

quantitative analysis because it can vary from

sample to sample.

To solve this problem one approach is

to post column infusion of an analyte into the

MS detector. The purpose of post column

infusion with the analyte is to raise the

background level so that the suppressionmatrix will show as negative peak. Any

deviation from the baseline caused by

injecting the matrix blank indicates the

existing of matrix effect.

Similarly, the recovery is determined

by comparing the MS response of extracted

sample with those spiked (post extraction) into

a blank matrix. Because both samples have the

matrix ingredients present, the matrix effect

can be considered the same for extracted

sample and post extracted sample. Any

difference in response now could be

considered caused by extraction recovery.

Chromatographic condition

determinationAnother important aspects with LC-

MS/MS use for bioanalysis of drug and

metabolites are determination of exact

chromatographic conditions including

selection of column, flow conditions,

detection system probe position, mode of

pump, injection volume etc. For Example:

Poor analyte column retention may result in

detrimental matrix effect that has been

identified as one of the major reasons why

bioanalytical LC-MS/MS method fails.

Conclusion

In order to determine the accurate

concentrations of drug and metabolites in

biological matrix by using LC-MS/MS,

there are many challenges. The major

challenges are, establishment of suitable

sample preparation technique, setting of

instrumental parameters such as, flow rate,

probe position, injection volume, mobile

phase determination etc. But at the same

time, we should be careful for the systemalso as the impurity of sample (For example;

some time sample prepared by the protein

precipitation technique remain with large

particle size can chock the column and

increases pressure of the system), pH of the


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mobile phase (very low pH solvent may

degrade the column) etc. may harm the

system.

References

1. Food and Drug Administration. (Draft)Guidance for Industry: BA and BE Studies

for Orally Administered Drug Products-

General Considerations Rock-ville, MD:

US Department of Health and Human

Services, Food and Drug Administration,

Center for Drug Evaluation and Research;

1999., pp1-14.

2. Guidelines for bioavailabity andbioequivalence studies, central drug

standard control organisation, directorate

general of health services ministry of

health and family walfare, govt. of India,

new delhi, march 2005, pp.1-34s.

3. Preliminary Draft Guidance for Industryon in-vivo Bioequivalence Studies Based

on Population and Individual

Bioequivalence Approaches: Availability,

The Food and Drug Administration

(FDA), 1997, (62), pp 249.

4. Ran Yin, Xiaohui Chen, Fei Han, et.al;LC-MS Determination and

Pharmacokinetic Study of Luteolin-7- O -

-D -glucoside in Rat Plasma after

Administration of the Traditional Chinese

Medicinal Preparation Kudiezi Injection

Chromatographia, 2008 (67), pp.961-965.

5. Liquid Chromatography MassSpectrometry Applications in

Agricultural, Pharmaceutical and

Environmental Chemistry, (M.A.

Brown, ed.) ACS Syrnp. Ser.,

Washington (1990), 1914-1926.

6. Wieling J., LC-MS-MS experienceswith internal standards

Chromatographia 2002, 55, pp. S.107-

113.

7. Breda, M. Marrari, R. Pianezzola, E.Strolin Benedetti, M., Determination of

ethambutol in human plasma and urine

by high-performance liquidchromatography with fluorescence

detectionJournal of Chromatography A,

1996, 729(1-2), pp. 301-307.

8. Henk A Characterization of stationaryphases for reversed-phase liquid

chromatography: column testing,

classification and chemical stability

Technische Universiteit Eindhoven,

1999, 7-8.

9. Siri, William, "Mass spectroscope foranalysis in the low-mass range", Review

of Scientific Instruments, 1947, 18 (8):

pp. 540545.

10.Weinmann W.; Gergov M.; Goerner M.MS/MS-libraries with triple

quadrupole-tandem mass spectrometers

for drug identification and drug


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screening: Structure elucidation by LC-

MS 2000 (20), pp. 934- 941.

11.Guidance for Industry, BioanalyticalMethod Validation U.S. Department of

Health and Human Services, Food and

Drug Administration, Center for Drug

Evaluation and Research (CDER), Center

for Veterinary Medicine (CVM), May

2001, BP., pp1-25.

12.Viswanathan CT, Bansal S, Booth B, et.al.; Workshop/Conference Report -

Quantitative Bioanalytical Methods

Validation and Implementation: Best

Practices for Chromatographic and

Ligand Binding Assays AAPS Journal,

2007; 9.

13.Vladimr Capka, and Spencer J. CarterMinimizing matrix effects in the

development of a method for the

determination of salmeterol in human

plasma by LC/MS/MS at low pg/mL

concentration levels Journal of

Chromatography B,

2007 (856), pp 285-293.


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Source of support: Nil, Conflict of interest: None Declared

Trouble Shooting During Bio Analytical Estimation of Drug and Metabolites Using LC-MSMS a Review[1]

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