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REVIEW ARTICLE
Year : 2011 | Volume : 2
| Issue : 1 | Page : 21-25
Detection limit
limit of detection
limit of quantitation
quantitation limit
methods for determination
of LOD and LOQ
Methods for the determination of limit of detection and limit of quantitation of the
analytical methods
Alankar Shrivastava, Vipin B Gupta
Department of Pharmaceutical Analysis, B.
R. Nahata College of Pharmacy, Mhow Neemuch Road, Mandsaur, Madhya Pradesh - 458 001, India
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Correspondence Address:
Alankar Shrivastava
Department of Pharmaceutical Analysis, BR Nahata College of Pharmacy, Mhow-Neemuch Road, Mandsaur, Madhya Pradesh 458 001
India
In this article
Abstract
Introduction
Detection and Qu...
Conclusion
Source of Support: None, Conflict of Interest: None
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DOI:10.4103/2229-5186.79345
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The
quality of an analytical method developed is always appraised in terms of suitability for its intended purpose, recovery, requirement for
standardization, sensitivity, analyte stability, ease of analysis, skill
subset required, time and cost in that order. It is highly imperative to establish
through a systematic process that the analytical method under question is acceptable for its intended purpose. Limit of detection (LOD) and limit
of quantification (LOQ) are two important performance characteristics in method validation. LOD and LOQ are terms used to describe the
smallest concentration of an analyte that can be reliably measured by an analytical procedure. There has often been a lack of agreement within
the clinical laboratory field as to the terminology best suited to describe this parameter. Likewise, there have
been various methods for estimating
it. The presented review provides information relating to the calculation of the limit of detection and limit of quantitation. Brief information about
differences in various regulatory agencies about these parameters is also presented here.
Keywords:Detection limit, limit of detection, limit of quantitation, quantitation limit, methods for determination of
LOD and LOQ
How to cite this article:

Shrivastava
A, Gupta VB. Methods for the determination of limit of detection and limit of quantitation of the analytical methods. Chron Young
Sci 2011;2:21-5
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Shrivastava
A, Gupta VB. Methods for the determination of limit of detection and limit of quantitation of the analytical methods. Chron Young
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http://www.cysonline.org/article.asp?issn=2229-5186;year=2011;volume=2;issue=1;spage=21;epage=25;aulast=Shrivastava[7/16/2016 11:57:02 AM]
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Methods for the determination of limit of detection and limit of quantitation of the analytical methods Shrivastava A, Gupta VB - Chron Young Sci
Analytical
method development and validation procedures are vital in the discovery
and development of drugs and pharmaceuticals. Analytical
methods are used to aid in the process of drug synthesis, screen potential drug candidates, support formulation studies, monitor the stability of
bulk pharmaceuticals and formulated products, and test final products for release. The quality of analytical data is a key factor in the success of a
drug and formulation development program. During the post approval commercial production stage of bulk drugs and pharmaceutical products,
the official or in-house test methods that have resulted from the analytical method development and validation process cycle become
indispensable for reliable monitoring of the integrity, purity, quality,
strength and potency of the manufactured products. There is often a need to
transfer methodology from one laboratory to another and/or to include it in official compendia. Such exercises include the use of a method by
large numbers of people, in various laboratories across the globe and on instruments manufactured by different manufacturers, thereby causing
a greater probability of decreased reproducibility and reliability. These problems can be foreseen and avoided by thorough validation of the
analytical method. [1]
Limit
of detection (LOD) and limit of quantitation (LOQ) parameters are related but have distinct definitions and should not be confused. The
intent is to define the smallest concentration of analyte that can be detected with no guarantee about the bias or imprecision of the result by an
assay, the concentration at which quantitation
as defined by bias and precision goals is feasible, and finally the concentration at which the
analyte can be quantitated with a linear response. [2] Comparison of regulatory authorities such as United States Pharmacopoeia (USP), [3]
Foods and Drugs Administration (FDA), [4] International Union of Pure and Applied Chemistry (IUPAC), [5] International Conference on
Harmonisation (ICH) [6] and Association of Analytical Communities (AOAC) [7],[8] for limit of detection and limit of quantitation are produced in
[Table 1] and [Table 2], respectively.
Table 1: Comparison of different guidelines for ''detection limit'' parameter of analytical method
validation[1]
Click here to view
Table 2: Comparison of different guidelines for 'quantitation limit' parameter of analytical method
validation[1]
Click here to view
Detection and Quantitation Limits (LOD and LOQ)
There
are several terms that have been used to define LOD and LOQ. In general, the LOD is taken as the lowest concentration of an analyte in a
sample that can be detected, but not necessarily quantified, under the stated conditions of the test. The LOQ is the lowest concentration of an
analyte in a sample that can be determined with acceptable precision and accuracy under the stated conditions of test. [9]
Although
reagent package inserts may state that an assay has a dynamic range that extends from zero concentration to some upper limit,
typically an assay is simply not capable of accurately measuring analyte concentrations down to zero. Sufficient analyte concentration must be
present to produce an analytical signal that can reliably be distinguished from "analytical noise," the signal produced in the absence of analyte.
[10]
However, some common methods [11] for the estimation of detection and quantitation limit are
Visual definition
Calculation from the signal-to-noise ratio (DL and QL correspond to 3 or 2 and 10 times the noise level, respectively)
Calculation from the standard deviation of the blank
Calculation from the calibration line at low concentrations
Where
F: Factor of 3.3 and 10 for DL and QL, respectively
SD:
Standard deviation of the blank, standard deviation of the ordinate intercept, or residual standard deviation of the linear regression
b: Slope of the regression line
The
estimated limits should be verified by analyzing a suitable number of samples containing the analyte at the corresponding concentrations.
The DL or QL and the procedure used for determination, as well as relevant chromatograms, should be reported.
Signal- to-noise
By using the signal-to-noise method, the peak-to-peak noise around the analyte retention time is measured, and subsequently, the concentration
of the analyte that would yield a signal equal to certain value of noise
to signal ratio is estimated. The noise magnitude can be measured either
manually on the chromatogram printout or by auto-integrator of the instrument. A signal-to-noise ratio (S/N) of three is generally accepted for
estimating LOD and signal-to-noise ratio of ten is used for
estimating LOQ. This method is commonly applied to analytical methods that exhibit
baseline noise. [11]
For
chromatography a test sample with the analyte at the level at which detection is required or determined is chromatographed over a period of
time equivalent to 20 times the peak width at half-height [Figure 1]. The signal-to-noise ratio is calculated from Equation (1).
Figure 1: Signal-to-noise examples of 10:1 (top) and 3:1 (bottom), using the method of the EP
Click here to view
where
H is the height of the peak, corresponding to the component concerned, in the chromatogram obtained with the prescribed reference
solution, and
measured from the maximum of the peak to the extrapolated baseline of the signal observed over a distance equal to 20 times the
width at half-height h is the peak-to-peak background noise in a chromatogram obtained after injection or application of a blank, observed over a
distance equal to 20 times the width at half-height of the peak in the chromatogram obtained.
This approach is specified in the European Pharmacopoeia. [5] It is important that the system is free from significant baseline drift and/or shifts
during this determination.
[Figure 1]
shows examples of S/N ratios of 10:1 and 3:1 which approximate the requirements for the QL and DL, respectively. This approach
works only for peak height measurements.
Blank determination
It is assumed that they both have the same variance and are normally distributed. As the curves overlap there is a probability that we could
conclude that we have detected the analyte when this is in fact due to the blank signal (false positive, error or type 1 error). Alternatively, we
can conclude that the analyte is not detected when it is in fact present (false negative, error or type 2 error). When addressing the issue about
when an analyte has been detected it is always a matter of risk. [11]
The
blank determination is applied when the blank analysis gives results with a nonzero standard deviation. LOD is expressed as the analyte
concentration corresponding to the sample blank value plus three standard deviation and LOQ is the analyte concentration corresponding to
the
sample blank value plus ten standard deviations as shown in the following equations:
LOD=Xb1 +3Sb1 ,
LOQ=Xb1 +10Sb1 ,
where Xb1 is the mean concentration of the blank and Sb1
is the standard deviation of the blank. This is a simple and quick method. The
weakness is that there is no objective evidence to prove that a low concentration of analyte will indeed produce a signal distinguishable from a
blank (zero concentration) sample. [9]
Linear regression
For a linear calibration curve, it is assumed that the instrument response y is linearly related to the standard concentration x for a limited range of
concentration. [9] It can be expressed in a model such as
y=a+bx.
This model is used to compute the sensitivity b and the LOD and LOQ. Therefore, the LOD and LOQ can be expressed as
LOD=3S a/b,
LOQ=10S a/b,
where S a
is the standard deviation of the response and b is the slope of the calibration curve. The standard deviation of the response can be
estimated by the standard deviation of either y-residuals, or y-intercepts,
of regression lines. This method can be applied in all cases, and it is
most applicable when the analysis method does not involve background noise. It uses a range of low values close to zero for calibration curve,
and with a more homogeneous distribution will result in a more relevant assessment.
Limit of blank
LoB as the highest apparent analyte concentration expected to be found when replicates of a sample containing no analyte are tested. Note that
although the samples tested to define LoB are devoid of analyte, a blank
(zero) sample can produce an analytical signal that might otherwise be
consistent with a low concentration of analyte. LoB is estimated by measuring replicates of a blank sample and calculating the mean result and
the standard deviation (SD). [2]
LoB=mean blank +1.645(SD blank )
After calculating this value LOD can be calculated according to LOD=LOB+1.645(SD low concentration sample ).
Precision-based approaches
The quantitation limit can also be obtained from precision studies. [10],[11]
For this approach, decreasing analyte concentrations are analyzed
repeatedly and the relative standard deviation is plotted against the corresponding concentration (precision function). If a predefined limit is
exceeded (such as 10% or 20%), the corresponding concentration is established as the quantitation limit However, in practice, due to the high
variability of standard deviations the true precision function is much more difficult to draw unless a large number of concentrations is included.
The QL can be specifically calculated [11]
using the actual precision of the analytical procedure at this concentration. The calculation is based on
the compatibility between analytical variability and specification acceptance limits. QL can be regarded as the maximum true impurity content of
the manufactured batch,
i.e., as the basic limit
AL Acceptance limit of the specification for the impurity.

s Precision standard deviation at QL, preferably under intermediate or reproducibility conditions. AL and s must have the same unit (e.g.,
percentage with respect to active, mg, mg/ml, etc.).
Nassay
Number of repeated, independent determinations in routine analyses, as far as the mean is the reportable result, i.e., is compared with
the acceptance limits. If each individual determination is defined as the reportable result, n=1 has to be used.
tdf Student t-factor for the degrees of freedom during determination of the precision, usually at 95% level of statistical confidence.
Conclusion
In
this review, the authors have tried to give information to the researchers engaged in establishing analytical profiles of the drug substances or
products. Data are adequate and sufficient to meet the laboratory's method requirements. The laboratory must be able to match the performance
data as described in the standard and to establish these
parameters a manufacturer would test a large number of sample replicates to increase
the robustness and the statistical confidence of the estimate. Comparison of all of the validation parameters in different regulatory agencies is
summarized by Chandran and Singh [1] and is a better option for curious readers.
References
Chandran
S, Singh RS. Comparison of various international guidelines for analytical method validation. Pharmazie 2007;62:4-14.
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David
A, Armbruster TP. Limit of Blank, Limit of Detection and Limit of Quantitation. Clin Biochem Rev 2008;29:S49-52.
Sanagi
MM, Ling SL, Nasir Z, Hermawan D, Ibrahim WA, Abu Naim A. Comparison of
Signal-to-noise, Blank Determination, and Linear
Regression Methods for
the Estimation of Detection and Quantification Limits for Volatile Organic Compounds by Gas Chromatography. J
AOAC Int 2009;92:1833-8.
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Putheti
RR, Okigbo RN, Patil SC, Advanapu MS, Leburu R. Method Development and Validations: Characterization of Critical Elements in
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European Pharmacopoeia. European Directorate for the Quality of Medicines. Strasbourg; 2007.
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Figures
[Figure 1]
Tables
[Table 1], [Table 2]
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A highly selective, sensitive and reversible fluorescence chemosensor for Zn2+and its cell viability
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Comparative
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High-performance liquid chromatography as a technique to determine protein adsorption onto

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An improved HPLC method for single-run analysis of the spectrum of hop bittering compounds usually
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Determination of 3,4-methylenedioxypyrovalerone (MDPV) in oral and nasal fluids by ion mobility spectrometry
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Ecological
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Electrochemical non-enzyme sensor for detecting clenbuterol (CLB) based on MoS2-Au-PEI-hemin layered
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Determination of high-sensitivity cardiac troponin I 99th percentile upper reference limits in a healthy Korean
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A carbon nanofiber-based label free immunosensor for high sensitive detection of recombinant bovine
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Solid lipid nanoparticles loaded with lipoylmemantine codrug: Preparation and characterization
Sara
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New1H NMR-Based Technique To Determine Epoxide Concentrations in Oxidized Oil

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Comparative pharmacokinetics of enrofloxacin, danofloxacin and marbofloxacin following intramuscular

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Determination of heavy metals in marine sediments using MAME-GFAAS

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Analysis and optimization of a hydrogel matrix for the development of a sandwich-type glucose biosensor
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Role of the anionic dopant of poly(3,4-ethylenedioxythiophene) for the electroanalytical performance:

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Polymethacrylate coated electrospun PHB Fibers: An exquisite outlook for fabrication of paper-based
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A novel approach for application of nylon membranes in the biosensing domain

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Azo dye functionalized graphene nanoplatelets for selective detection of bisphenol A and hydrogen peroxide
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Improved quantitative analysis of molecular constituents of wastewater sludge pellets using double-shot
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A New Approach for Quantitative Determination of ?-Cyclodextrin in Aqueous Solutions: Application in

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Biological imaging without autofluorescence in the second near-infrared region

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A candidate reference method using ICP-MS for sweat chloride quantification

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Advances
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Comparison
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Volatile organic compounds generated by cultures of bacteria and viruses associated with respiratory
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2-Acetyl-1-Pyrroline
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Simultaneous
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A novel method for quantification of human hemoglobin from dried blood spots by use of tandem mass
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The simultaneous detection and quantification of p-aminobenzoic acid and its

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metabolites in human urine using LCMS/MS
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Reduced
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Reduced
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Discrimination
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Enhancement of performance in porous bead-based microchip sensors: effects of chip geometry on bio-agent
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HPLC method for urinary theobromine determination: Effect of consumption of cocoa products on theobromine
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Extraction-spectrophotometric
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Extractive
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Carboxylated,
Fe-Filled Multiwalled Carbon Nanotubes as Versatile Catalysts for O2Reduction and H2Evolution
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Sol-Gel Deposition of Iridium Oxide for Biomedical Micro-Devices

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ESTIMATION OF THE WITHDRAWAL PERIOD FOR AMOXICILLIN IN POMPANO (TRACHINOTUS BLOCHII)

SERUM AND MUSCLE
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Development
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Microsphere integrated microfluidic disk: synergy of two techniques for rapid and ultrasensitive dengue
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Samira
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NORM in the East Midlands oil and gas producing region of the UK
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Evaluation
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Growth of healthy and sanitizer-injured Salmonella cells on mung bean sprouts in different commercial
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Assessment of tandem mass spectrometry and high-resolution mass spectrometry for the analysis of
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Fast, sensitive and selective colorimetric gold bioassay for dopamine detection
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Characteristic investigation of scanning surface plasmon microscopy for nucleotide functionalized nanoarray
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ZnO oxide films for ultrasensitive, rapid, and label-free detection of neopterin by surface-enhanced Raman
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EVALUATION OF AMOXICILLIN WITHDRAWAL PERIOD TIME IN POMPANO LIVER AND KIDNEY

Chia-Yih Wang,Shi-Yuan Sheu,Yi-Chih Lei,Jiann-Hsiung Wang,Ming-Huang Chang,Pay-Heng Chen,Tong-Hsuan Chang,Tzong-Fu Kuo
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Changes
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Development of a mobile tracer correlation method for assessment of air emissions from landfills and other
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Determination of total mercury in fish and sea products by direct thermal decomposition atomic absorption
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Aging effect and antibody immobilization on COOH exposed surfaces designed for dengue virus detection
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Development
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Label-free detection of small-molecule binding to a GPCR in the membrane environment

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Evaluation
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Epicocconone,
a sensitive and specific fluorescent dye for in situ quantification of extracellular proteins within
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Surface enhanced Raman scattering-active worm-like Ag clusters for sensitive and selective detection of
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Determination of environmental safety level with laser-induced breakdown spectroscopy technique

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Sensitive
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Simultaneous
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Roberta Ottria,Alessandro Ravelli,Fausto Gigli,Pierangela Ciuffreda
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Derivatization
Method for Sensitive Determination of 3-Hydroxybenzo[a]pyrene in Human Urine by Liquid
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Detection of methicillin-resistant Staphylococcus aureus using novel lytic phage-based magnetoelastic

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Applying
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Detection of Hepatitis B virus antigen from human blood: SERS immunoassay in a microfluidic system
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Novel and sensitive reversed-phase high-pressure liquid chromatography method with electrochemical
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Development
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Robust and versatile ionic liquid microarrays achieved by microcontact printing

Christian A. Gunawan,Mengchen Ge,Chuan Zhao
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Quantification of Halobetasol Propionate and Its Impurities Present in Topical Dosage Forms by StabilityIndicating LC Method
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Toxicokinetic of flumethrin in rabbits

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A versatile-deployable bacterial detection system for food and environmental safety based on LabTubeautomated DNA purification, LabReader-integrated amplification, readout and analysis
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Optimization
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A Facile and Sensitive Method for Quantification of Cyclic Nucleotide Monophosphates in Mammalian Organs:
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Liquid chromatographic determination of CPZEN-45, a novel anti-tubercular drug, in biological samples

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A Rapid, Isocratic HPLC Method for Determination of Insulin and Its Degradation Product
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A novel organicinorganic hybrid conducting copolymer for mediated biosensor applications

Tugba Soganci,Dilek Odaci Demirkol,Metin Ak,Suna Timur
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Geochemistry of magnetite from porphyry Cu and skarn deposits in the southwestern United States
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Synthesis
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Isoflavone
Content and Profile Comparisons of Cooked SoybeanRice Mixtures: Electric Rice Cooker versus
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Highly
Selective Mercury Detection at Partially Oxidized Graphene/Poly(3,4Ethylenedioxythiophene):Poly(Styrenesulfonate) Nanocomposite Film-Modified Electrode
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Optical detection of amine vapors using ZnTriad porphyrin thin films

Javier
Roales,Jos M. Pedrosa,Mara G. Guilln,Tnia Lopes-Costa,Sara M.A. Pinto,Mrio J.F.
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A Rapid Phytohormone and Phytoalexin Screening Method for Physiological Phenotyping

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Scavenging
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A new paper-based platform technology for point-of-care diagnostics

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A novel ion-exclusion chromatographymass spectrometry method to measure concentrations and cycling

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Label-free detection of lysozyme in wines using an aptamer based biosensor and SPR detection
Iuliana Mihai,Alis Vezeanu,Cristina Polonschii,Camelia Albu,Gabriel-Lucian Radu,Alina Vasilescu

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Carbon
dots as nanoantennas for anti-inflammatory drug analysis using surface-assisted laser
desorption/ionization time-of-flight mass spectrometry in serum
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Phenolic
Compounds Involved in Grafting Incompatibility ofVitisspp: Development and Validation of an
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Polarographic Determination of Sodium Hydrosulfite Residue (Dithionite) in Sugar and Loaf Sugar
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Iminodiacetic acid functionalized polypyrrole modified electrode as Pb(II) sensor: Synthesis and DPASV
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Methods For The Determination of Limit of Detection and Limit of Quantitation of

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Detection and Quantitation Limits (LOD and LOQ)

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http://www.cysonline.org/article.asp?issn=2229-5186;year=2011;volume=2;issue=1;spage=21;epage=25;aulast=Shrivastava[7/16/2016 11:57:02 AM]

AL Acceptance limit of the specification for the impurity.

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[Table 1], [Table 2]

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