International Journal of Trend in Scientific

Research and Development (IJTSRD)

International Open Access Journal
ISSN No: 2456 - 6470 | www.ijtsrd.com | Volume - 2 | Issue – 4

A General Review oon Bioanalytical Method

ethod Development &
Validation ffor LC-MS/MS
Ashutosh Badola, Preeti Joshi, Preeti Kothiyal
Shri Guru Ram Rai Institute of Technology and Sciences, Dehradun
Dehradun, Uttarakhand,
Uttarakhand India

ABSTRACT
Rapid growth in the use of LC-MS/MS MS/MS for the interpretation of pharmacokinetic, bioavailability,
bioavailabi
analysis of drugs in biological matrices has been drug-drug
drug interaction, bioequivalence and
compelled by the need for timely and high high-quality compatibility studies [1]. Validation of any analytical
data at every stages in drug discovery and method ensures that the developed method is
development process: from throughput screening of reproducible, stable, sensitive, robust, suitable and
drug candidates and rapid data generation for pre pre- reliable for its application in blood, plasma, urine,
clinical studies to almost 'real-time'
time' analysis of serum and faeces analysis. Bioanalytical validation
clinical samples. A well developed bioanalytical ensures the high-quality
quality data for regulatory
development and its validation plays
ays a pivotal role in submission as well as for the drug discovery and
achieving the goals. . The aim behind this review is development [2].
to enlighten the need of validation which provide a
practical approach for determining the different The official test methods that result from these
parameters like selectivity, specifity, limit of processes are used by quality control laboratories
labo to
detection, lower limit of quantitation,
ntitation, linearity, range, ensure the identity, purity, potency and performance
accuracy, precision, recovery, stability, ruggedness, of drug products [3] and includes all the procedures
and robustness to help the perfect studies of demonstrating particular method used for quantitative
pharmacokinetic, toxic kinetic, bioavailability and measurement of analytes in a given biological matrix,
bioequivalence. Bio-analysis
analysis study is for the such as blood, plasma, serum, or urine, reliable and
quantitative determination of drug and their reproducible for the intended use [4,5]. The analysis
metabolites in biological fluids. Accurate and robust thus carried out must be verified for its alleged
methods for quantitative analysis of drug and their purpose and must be validated. An investigation
metabolites are important for the successful conduct should be performed during each step to determine
of pre-clinical, bio-pharmaceutics
pharmaceutics and clinical whether the external environment,
environmen matrix or
pharmacology. procedural variables can affect the estimation of
analyte in the matrix from the time of collection up to
Keywords - LC-MS/MS
MS/MS bioanalysis , Bio
Bio-analytical the time of analysis [6]. Recent progress in methods
method development,Validation parameters, sample development has been largely a result of
Preparation LLE, SPE. improvements in analytical instrumentation.

INTRODUCTION Both HPLC and LC-MS/MS MS/MS can be used for the
The word “high Bioanalytics” refers to the analysis of bioanalysis of drugs in plasma. Each of the
the desired analyte in biological fluids. In the present instruments has its own merits and demerits. HPLC
pharmaceutical industry,
try, the bioanalytical methods coupled with UV, PDA or fluorescence detector can
are playing a crucial role in the quantitative be used for estimation of many compounds but it does
determination of low molecular weight drug not give the high sensitivity as required by some of
molecules and macromolecules. The quantitative the potent, low dose drugs and lacks selectivity [7].
determination leads to the evaluation and The main advantages of LCMS-MS
LCMS include low

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 International Journal of Trend in Scientific Research and Development (IJTSRD) ISSN: 2456-6470
detection limits, the ability to generate structural chromatographic system. General procedures for
information, the requirement of minimal sample sample preparation like liquid/liquid extraction, solid-
treatment and the possibility to cover a wide range of phase extraction (SPE) and protein precipitation.
analytes differing in their polarities. Despite their high
sensitivity and selectivity LC/MS/MS instruments are Liquid-liquid extraction- It is based on the principle
limited to some extent due to matrix-induced of selective extraction of intended analyte present in
differences in ionization efficiencies and ion liquid sample through immiscible organic solvent.
suppression/enhancement effects due to biological LLE is based on differential solubility and
matrix. HPLC coupled with UV, PDA or fluorescence partitioning of two immiscible liquid phases in one, it
detector offers a cost effective bioanalytical method required two immiscible phases, in most of the cases
[8,9,10]
. Depending on the sensitivity, selectivity and one aqueous and second organic phase, both phases
cost effectiveness of the method a choice needs to be must be immscible [15]. Analyte can be removed from
made between HPLC AND LCMS-MS. the matrix selectively by choosing a suitable
extraction solvent and buffering ( pH adjustment of
METHOD DEVELOPMENT the sample to analyte in unionized form)of sample, if
Analytical method development is the process of required. The solvents should match the analytes
creating a procedure to enable a compound of interest polarity while still being immiscible with water and it
to be identified and quantified in a matrix. A should preferably be compatible with the detection
compound can often be measured by several methods method. A large volume of the extraction solvent,
and the choice of analytical method involves many compared to the sample, favours partitioning and the
considerations, such as: chemical properties of the enrichment of the analytes is often insufficient [16,17].
analyte, concentrations levels, sample matrix, cost of The solvents which generally used are tert-butyl
the analysis, speed of the analysis, quantitative or methyl ether(TBME), dichloromethane(DCM), ethyl
qualitative measurement, precision required and acetate( EA), diethyl ether( DEE), hexane etc or in
necessary equipment [11,12]. The analytical chain combination with any other suitable solvent can be
describes the process of method development and used as an extraction solvent.
includes sampling, sample preparation, separation,
detection and evaluation of the results.
Solid-phase extraction- It is based on adsorption (or)
Partitioning on to solid sorbent (Absorbent)selective
Sample preparation & extraction procedures for
retardation of analyte using solid sorbent under
drug and its metabolites from biological samples
specific conditions. SPE based on the selective
The biological media that contain the analyte are
adsorption mechanism. If the targeted analyte are
usually blood, plasma, urine, serum etc. Blood is
adsorbed on the solid phase, they can selectively be
usually collected from by different blood withdrawl
removed or eluted by using an appropriate elution
routes like retro-orbital sinus, tail vein method, dorsal
solvent. SPE occur between a solid phase and a liquid
pedal vein (depending on the assay sensitivity and the
phase. SPE is more efficient separation process than
total number of samples taken for a study being
LLE. It is easier to obtain a higher recovery of
performed). The blood is directly withdrawn into
analyte. It employs a small plastic disposable column
tubes with an anticoagulant, e.g. EDTA, heparin etc.
or cartridge, often the barrel of a medical syringe
Plasma is obtained by centrifugation at 4000 rpm for
packed with 0.1 to 0.5g o sorbent. The sorbent is
15 min. About 30 to 50% of the original volume is
usually reversed phase SPE (RP-SPE) assembles both
collected [13].
LLE and reversed phase HPLC in its separation
characterstics [17]. In SPE, liquid sample is added to
The purpose of sample preparation is to clean up the
the cartridge and wash solvent is selected so that the
sample before analysis and/or to concentrate the
analyte is either stongly retained (k>>1) or unretained
sample. Material in biological samples that can
(k=0). When the analyte is strongly retained,
interfere with analysis, the chromatographic column
interferences are eluted or washed from the cartridge
or the detector includes proteins, salts, endogenous
so as to minimize their presence in the final analyte
macromolecules, small molecules and metabolic
fraction. The analyte is then eluted in a small volume
byproducts [14]. A goal with the sample preparation is
with strong elution solvent, collected, and either
also to exchange the analyte from the biological
ijected directly or evaporated to dryness followed by
matrix into a solvent suitable for injection into the
dissolution in the mobile phase. In the opposite case,

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 International Journal of Trend in Scientific Research and Development (IJTSRD) ISSN: 2456-6470
where analyte is weakly retained, interferences are reproducible methods and techniques are always very
strongly held on the cartridge and the analyte is demanding for the drugs and its metabolites studies
collected for the further treatment [18]. for bioavailability (BA), bioequivalence (BE) and
pharmacokinetic (PK) parameter for conducting the
Protein precipitation- Protein precipitation is the
pre clinical studies [20-21]. A perfect approach to reach
simple method of extraction as compared to the LLE
the drugs and its metabolites best efficacy and side
and SPE. This can be carried out by using the suitable
effects value can be known by the accurate and
organic solvents which has good solubility of the
sensitive method development and validation. In
analyte and protein precipitating properties.
respect of economy and market demand the chief and
Acetonitrile is the first choice of solvent for protein
best bio-analytical methods are adopted for routine
precipitation due to its complete precipitation of
analysis [22]. In bio-analytical method validation
proteins and methanol is the second choice of organic
different types and levels are come which must be
precipitant provided the solubility of the analyte in
need to understand basic requirement in the process.
these solvents. After protein precipitation the
Here all types are defined in very specific manner.
supernatant obtained can be injected directly in to the
HPLC or it can be evaporated and reconstituted with
Full Validation- Full validation of bioanalytical
the mobile phase [19].
methods is important:
 During development and implementation of a
novel bioanalytical method.
 For analysis of a new drug entity.
 For revisions to an existing method that add
metabolite quantification.

Partial Validation- Partial validations evaluate

modifications of already validated bioanalytical
methods. Partial validation can range from as little as
one intra-assay accuracy and precision determination
to a nearly full validation. Typical bioanalytical
method modifications or changes that fall into this
Fig 1: General steps of method development & category include but are not limited to:
validation  Bioanalytical method transfers between
laboratories or analysts.
BIOANALYTICAL METHOD VALIDATION  Change in analytical methodology (e.g.,
Bioanalytical method validation includes all of the change in detection systems).
procedures required to demonstrate that a particular  Change in anticoagulant in harvesting
bioanalytical method for the quantitative biological fluid (e.g., heparin to EDTA).
determination of the concentration of an analyte (or  Change in matrix within species (e.g., human
series of analytes) in a particular biological matrix is plasma to human urine).
reliable for the intended application. The most widely  Change in sample processing procedures.
employed bioanalytical techniques include , mass  Change in species within matrix (e.g., rat
spectrometry based methods (such as GC-MS and plasma to mouse plasma).
LC-MS), tandem mass spectrometry based methods  Change in relevant concentration range.
(such as LC-MS-MS) and ligand- based assays (such
as RIA and ELISA). Many of the principles, Cross-Validation- Cross-validation is a comparison
procedures, and requirements of bioanalytical method of validation parameters when two or more
validation are common to all types of analytical bioanalytical methods are used to generate data
methodologies. within the same study or across different studies. An
example of cross-validation would be a situation in
Bio-analytical method validation or bio-analysis of which an original validated bioanalytical method
drug has its own value which will depend upon serves as the reference, and the revised bioanalytical
analytes nature and technology which use for the method is the comparator. The comparisons should
method development and validation. A reliable and be done both ways, when sample analyses within a

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 International Journal of Trend in Scientific Research and Development (IJTSRD) ISSN: 2456-6470
single study are conducted at more than one site or replicate analysis of samples containing known
more than one laboratory, cross-validation with amounts of the analyte (i.e., QCs). Accuracy should
spiked matrix standards and subject samples should be measured using a minimum of five determinations
be conducted at each site or laboratory to establish per concentration. A minimum of three concentrations
inter-laboratory reliability [23,24]. in the range of expected study sample concentrations
is recommended. The mean value should be within
Need & importance for analytical methods to be 15% of the nominal value except at LLOQ, where it
validated should not deviate by more than 20%. The deviation
The main objective of a method validation is to check of the mean from the nominal value serves as the
the reliability of a particular method for evaluating an measure of accuracy.
analyte concentration in a specific biological matrix,
such as blood, serum, plasma, urine, or saliva. Precision is defined as The closeness of individual
Moreover, if an anticoagulant is used, validation measures of an analyte when the procedure is applied
should be performed using the same anticoagulant as repeatedly to multiple aliquots of a single
for the study samples [25]. Generally a full validation homogeneous volume of biological matrix and it is
should be performed for each species and matrix measured by using a minimum of five determinations
concerned for the standardization o the procedure by per concentration. A minimum of three concentrations
which an analyte is analyzed. in the range of expected study sample concentrations
is recommended. The precision determined at each
PARAMETERS OF VALIDATION- [26-32] concentration level should not exceed 15% of the
coefficient of variation (CV) except for the LLOQ,
1. System suitability where it should not exceed 20% of the CV.
System suitability should be confirmed prior to each
run to ensure the performance of the instrument for
𝑚𝑒𝑎𝑠𝑢𝑟𝑒𝑑 𝑣𝑎𝑙𝑢𝑒 − 𝑡𝑟𝑢𝑒 𝑣𝑎𝑙𝑢𝑒
that particular bioanalytical method. The validity of % 𝐵𝑖𝑎𝑠 = × 100
the analysis is done on routinely basis and checked by 𝑡𝑟𝑢𝑒 𝑣𝑎𝑙𝑢𝑒
evaluation of calibration curves and QC sample in 𝑆𝐷
each analytical run. System performance experiment % 𝐶𝑉 = × 100
was performed by injecting sequence of injections at 𝑀𝑒𝑎𝑛
the beginning of analytical batch or before any re-
injection. The Signal to Noise ratio should be more 4. Calibration curve/Linearity range
than or equal to 5 for LLOQ QC sample. It represents the relationship between the response of
the instrument and the known concentration of the
2. Selectivity/specificity analyte. A calibration curve should be performed for
Selectivity is a term defined as the ability of an each analyte in the sample. The calibration standards
bioanalytical method to differentiate and quantify the can contain more than one analyte. A calibration
analyte in the presence of other components in the curve should be prepared in the same biological
sample & specificity is a term which is defined as the matrix as the samples in the intended study by spiking
ability of the bioanalytical method to produce a signal the matrix with known concentrations of the analyte.
only for the analyte of interest and not for other The calibration curves were constructed from a blank
interfering components. Generally, the procedure sample (a plasma sample processed without IS), a
followed for the evaluation of selectivity & specificity zero sample (a plasma processed with IS), and eight
is to compare the response of an analyte in the concentrations covering the expected range including
biological sample at the lower limit of quantification the LLOQ. The drug-to-IS peakarea ratio was plotted
(LLOQ) with blank matrix sample. It is recommended against the respective standard drug concentration to
to take blank matrix from at least six different sources obtain the graph and the linearity was evaluated by
and compare it with the spiked LLOQ in the matrix. weighted (1/x) least-squares regression analysis. The
acceptance criteria for each calculated standard
3. Accuracy & Precision concentration was no more than 15% deviation from
Accuracy is defined as the closeness of mean test the nominal value, except for the LLOQ, for which
results obtained by the method to the actual value 20% was acceptable. Unknown sample peak–area
(concentration) of the analyte and it is determined by

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 International Journal of Trend in Scientific Research and Development (IJTSRD) ISSN: 2456-6470
ratios were then interpolated from the calibration replicates per QC concentration level before and after
curve to provide concentrations of unknown samples. the stability storage. The acceptance criteria suggest
that the mean accuracy of the measurements at each
5. Recovery level should be within ±20% deviation of the
It is defined as the detector response obtained from an theoretical concentrations , while USFDA draft
amount of the analyte added to and extracted from the guidelines suggest that the stability study sample
biological matrix, compared to the detector response results should be within ±15% of the nominal
obtained for the true concentration of the analyte in concentrations.
solvent. The procedure involves by comparing the
analytical results for extracted samples at three 7. Matrix effect
concentrations (low, medium, and high) with It is defined as the effect or the response produced by
unextracted standards that represent 100% recovery. any undesired analyte which have an interference
Recovery of the analyte need not be 100%, but the with the analyte response. Generally, it is caused by
extent of recovery of an analyte and IS should be the matrix components or the metabolites which get
consistent, precise, and reproducible. eluted along with the analyte. Matrix effect can also
be caused when molecules co-eluting with the
Calculated concentration compounds of interest alter the ionization efficiency
Recovery % = × 100 of the electrospray interface. Usually, the matrix
𝑆𝑝𝑖𝑘𝑒𝑑 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛
effect is assessed either by post extraction addition
6. Stability method or the post-column infusion method.
Validation studies should determine the analyte Matrix effect can be evaluated by spiking analyte at
stability after the freeze-thaw cycles, short term and two concentration levels (low and high QC) in six
long-term storage. The stability of the analyte should different lots of blank matrix and reading against
be evaluated in the stock and working solutions using freshly prepared CC. The matrix effect is calculated
solutions at or near the highest and lowest by matrix factor (MF). Matrix factor can be calculated
concentration levels under the actual solution storage by the following formula:
conditions. According to EMA guidelines it is
recommend to evaluate the stability of at least 3

Matrix Effect = Analyte (or IS) response in spiked blank extract

Analyte (or IS) response in neat solution

8. Robustness

The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small, but
deliberate variations in method parameters and provides an indication of its reliability during normal usage.
The evaluation of robustness should be considered during the development phase and depends on the type of
procedure under study. It should show the reliability of an analysis with respect to deliberate variations in
method parameters.

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 International Journal of Trend in Scientific Research and Development (IJTSRD) ISSN: 2456-6470
2456

Bioanalytical
validation methods US FDA guidelines

Selectivity Analyses of blank samples of the appropriate biological matrix (plasma, urine or other
(specificity) matrix) should be obtained from at least six sources. Each blank should be tested for
interference and selectivity should be ensured at LLOQ

Accuracy Should be measured using a minimum of six determinations per concentration.

Minimum of three concentrations in the range of expected concentrations is
recommended for determination of accuracy. The mean should be ±15% of the actual
value except at LLOQ, wh where
ere it should not deviate by ±20%. This deviation of mean
from the true values serves as the measure of accuracy

Precision Precision should be measured using a minimum of five determinations per

concentration. Minimum of three concentrations in the ra
range
nge of expected concentrations
is recommended. The precision determined at each concentration level should not
exceed 15% of the CV except for the LLOQ, where it should not exceed 20% of the CV

Recovery Recovery experiments should be performed at thre threee concentrations (low, medium and
high) with unextracted standards that represent 100% recovery

Calibration curve Should consist of a blank sample (matrix sample processed without internal standard), a
zero sample (matrix sample processed with internal standard) and six to eight non-zero
non
samples covering the expected range, including LLOQ

LLOQ Analyte response shshould

ould be five times the response compared to blank response.
Analyte peak should be identifiable, discrete and reproducible with a precision of 20%
and an accuracy of 80
80–120%

Freeze–thaw stability Analyte stability should be determined after three freeze

freeze–thaw
thaw cycles. At least three
aliquots at each of the low and high concentrations should be stored at intended storage
temperature for 24 hours and thawed at room temperature. When completely thawed,
refreezee again for 12
12–24
24 hours under same conditions. This cycle should be repeated
two more times, then analyze on third cycle. Standard deviation of error should be
<15%. If analyte is unstable, freeze at -70°C for three freeze–thaw
thaw cycles

Short-term stability Three aliquots of each of the low and high concentrations should be thawed at room
temperature and kept at this temperature for 44–24
24 hours and analyzed. Percent deviation
should be <15%

Long-term stability At least three aliquots of each of low and high concentrations at same conditions as
study samples. Analyze on three separate occasions. Storage time should exceed the
time between the date of first sample collection and the date of last sample analysis

Stock-solution Stability of stock solutions of drug and the internal standard should be evaluated at
stability room temperature for at least 6 hours. Percent deviation should be <15%

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 International Journal of Trend in Scientific Research and Development (IJTSRD) ISSN: 2456-6470
QC samples QC samples in duplicates at three concentration levels (one near the 3× LLOQ, one in
mid range, one close to high end) should be incorporated at each assay run. At least four
out of every six should be within 15% of the respective nominal value. Two of the six
may be outside of 15% but not both at the same concentration. Minimum number QCs
should be at least 5% of total number of unknown samples or six total QCs, whichever
is greater

Table No. 1: US FDA guidelines for bioanalytical method validation

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