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Year : 2010  |  Volume : 1  |  Issue : 1  |  Page : 1-10 Table of Contents     

Trouble shooting during bioanalytical estimation of drug and metabolites using LC-MS/MS: A review

1 School of Pharmaceutical Sciences, Jaipur National University, Jaipur, Rajasthan, India
2 LBS College of Pharmacy, Tilak Nagar, Jaipur, Rajasthan, India
3 Maharishi Arvind Institute of Pharmacy, Mansarovar, Jaipur, Rajasthan, India

Date of Submission08-Jan-2010
Date of Decision11-Feb-2010
Date of Acceptance01-Mar-2010
Date of Web Publication2-Nov-2010

Correspondence Address:
Ganesh N Sharma
School of Pharmaceutical Sciences, Jaipur National University, Jaipur, Rajasthan
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Source of Support: None, Conflict of Interest: None

PMID: 22247825

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Bioanalysis 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.

Keywords: Bioanalysis, LC-MS/MS, Ion suppression, Matrix effect

How to cite this article:
Sharma GN, Singhal MM, Sharma K K, Sanadya J. Trouble shooting during bioanalytical estimation of drug and metabolites using LC-MS/MS: A review. J Adv Pharm Technol Res 2010;1:1-10

How to cite this URL:
Sharma GN, Singhal MM, Sharma K K, Sanadya J. Trouble shooting during bioanalytical estimation of drug and metabolites using LC-MS/MS: A review. J Adv Pharm Technol Res [serial online] 2010 [cited 2021 Oct 21];1:1-10. Available from: https://www.japtr.org/text.asp?2010/1/1/1/70512

   Introduction Top

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 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 Top

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 Top

  1. To identify and /or quantify illicit drugs in biological samples for the detection of substances of abuse
  2. To assay foreign substances in biological materials, sometime postmortem for evidence of poison in toxicological and forensic use.
  3. To analyze trace elements and toxic substances in natural science and environmental research.
  4. To monitor the concentration of drugs and metabolites in tissues, blood, plasma, serum and urine specimens for a better understanding of the pharmacology and toxicity of drugs.
  5. To apply therapeutic drug monitoring in the management of the drug treatment in patients.

   LC-MS/MS Top

Liquid chromatography and mass spectrometry is two segment of a LC­MS/MS system. The sample under investigation has to be introduced into the ionisation source of the instrument. Inside the ionisation source, sample molecules are 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 Top

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 Top

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.

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 available tandem 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, magnetic sector-quadrupole, and more recently, the quadruple-time-of-flight geometries. [10]

   Critical points in bioanalysis with LC-MS/MS Top

In order to determine the accurate concentration of drug and metabolites in biological matrix there are so many challenges. These challenges make the overall process difficult. The major challenges are as:

  • What would be the exact chromatographic 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 Top

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 interact with 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 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:

  1. Maximum plasma concentration (C max ) of the drug and metabolite: knowing C max helps the establishment of target sensitivity of method.
  2. Physiochemical properties of the drug and metabolite: following properties aid to guess and select proper extraction and sample preparation procedure.
  1. Molecular weight (MW)
  2. Solubility
  3. Structure
  4. Dissociation constant (pKa)
  5. Melting point.

   Determination of lower and upper limit of quantification Top

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 1/20 th of the C max 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 Top

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 Top

  1. Physiochemical property of drugs
  2. Selection of solvent for extraction: A good solvent contains following characteristics:
    • Drug should be soluble properly in the solvent.
    • It should not be flammable and hazardous.
    • It should be easily available and should not be of high cost.
    • It should be easily evaporated, so that drug can be re-obtained from the solution.
  3. Selection of medium (acidic/ basic or neutral)
  4. Selection of extraction procedure i.e. either liquid-liquid extraction (LLE), protein precipitation (PP), or solid phase extraction (SPE).

   Selection of plasma volume spiked Top

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;

   Selection of drug and metabolite volume to spike Top

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 μl, so the volume of drug and metabolite to be added will be 25μl of each.

   Selection of internal standard Top

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 Top

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 that could 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.

   Ion suppression Top

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 spectrometer's 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 suppression matrix 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 determination Top

Another important aspects with LC­MS/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 Top

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 system also 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 mobile phase (very low pH solvent may degrade the column) etc. may harm the system.[Figure 1],[Figure 2],[Figure 3]
Figure 1 :Components of an LC system.

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Figure 2 :Typical flow diagram of a Mass Spectrometer

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Figure 3 :Typical LC-MS/MS configuration

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   References Top

1.Food and Drug Administration. (Draft) Guidance for Industry: BA and BE Studies for Orally Administered Drug Products­General Considerations Rock-ville, MD: US Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research; 1999., pp1-14.  Back to cited text no. 1
2.Guidelines for bioavailabity and bioequivalence 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.  Back to cited text no. 2
3.Preliminary Draft Guidance for Industry on in-vivo Bioequivalence Studies Based on Population and Individual Bioequivalence Approaches: Availability, The Food and Drug Administration (FDA), 1997, (62), pp 249.  Back to cited text no. 3
4.Ran Yin, Xiaohui Chen, Fei Han, et.al; " LC-MS Determination and Pharmacokinetic Study of Luteolin-7- O - LI--D -glucoside in Rat Plasma after Administration of the Traditional Chinese Medicinal Preparation Kudiezi Injection" Chromatographia, 2008 (67), pp.961-965.  Back to cited text no. 4
5.Liquid Chromatography Mass Spectrometry Applications in Agricultural, Pharmaceutical and Environmental Chemistry, (M.A. Brown, ed.) ACS Syrnp. Ser., Washington (1990), 1914-1926.  Back to cited text no. 5
6.Wieling J., "LC-MS-MS experiences with internal standards" Chromatographia 2002, 55, pp. S.107­-113.  Back to cited text no. 6
7.Breda, M. Marrari, R. Pianezzola, E. Strolin Benedetti, M., Determination of ethambutol in human plasma and urine by high-performance liquid chromatography with fluorescence detection Journal of Chromatography A, 1996, 729(1-2), pp. 301-307.  Back to cited text no. 7
8.Henk A "Characterization of stationary phases for reversed-phase liquid chromatography: column testing, classification and chemical stability" Technische Universiteit Eindhoven, 1999, 7-8.  Back to cited text no. 8
9.Siri, William, "Mass spectroscope for analysis in the low-mass range", Review of Scientific Instruments, 1947, 18 (8): pp. 540-545.  Back to cited text no. 9
10.Weinmann W.; Gergov M.; Goerner M. "MS/MS-libraries with triple quadrupole-tandem mass spectrometers for drug identification and drug screening: Structure elucidation by LC­MS" 2000 (20), pp. 934- 941.  Back to cited text no. 10
11.Guidance for Industry, Bioanalytical Method 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.  Back to cited text no. 11
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.  Back to cited text no. 12
13.Vladimir Capka and Spencer J. Carter "Minimizing 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.  Back to cited text no. 13


  [Figure 1], [Figure 2], [Figure 3]

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