LOD Determination With Noiseless Detectors

1

Dr. Michael LouisJordi Labs

April 2, 2020

Limit of Detection

Introduction of noiseless detectors

Methods for analysis of LOD

Most practical methods for noiseless detection

Outline

2

Limits of Detection

3

• Limits of detection and quantitation are critical for analytical instrumentation and validation of analytical methods

• Necessary in determining toxicological thresholds for compounds of interest across a wide variety of industries including pharmaceutical, medical devices, food contact materials, as well as other consumer products

Detection and Quantitation Limits

4

Limit of Detection (LOD)• USP/International Conference on Harmonization (ICH)-Lowest amount of

analyte in the sample, which can be detected but not necessarily quantitated under stated experimental conditions

• US FDA-Not explicitly described

Limit of Quantitation (LOQ)• USP/International Conference on Harmonization (ICH)-Lowest amount of

analyte in the sample, which can be quantitatively determined with suitable precision and accuracy

• US FDA-The lowest amount of analyte that can be quantitatively determined with suitable precision and accuracy also called LLOQ (Lower Limit of Quantification)

Detection and Quantitation Limits

5

Limit of Detection/Quantitation (LOD/LOQ)• By visual evaluation• Based on S/N ratio (LOD-

3:1/LOQ-10:1)• Based on standard

deviation of response and slope (LOD-3.3σ/s-LOQ-10σ/s)

• Traditionally use of S/N has been used to determine LOD/LOQ

Triple Quadrupole (QqQ)

6

• First QqQ developed in late 1970’s

• Significantly improved detection limits for Gas Chromatography (GC), Liquid Chromatography (LC) and Inductively Coupled Plasma (ICP) analysis

• Use of tandem quads, allows for significant noise reduction increasing sensitivity of target analyte

• Little to no noise, required additional means for evaluating LOD

LCMS/GCMS QqQ

7

• LC-QqQ and GC-QqQ can both operate using multiple reaction monitoring (MRM) where parent ions are isolated in quad one then are further fragmented in the collision cell and product ions are selected in quad two significantly reducing background noise

Ionization

SourceQ1 Q2

Collision

CellDetector

ICP-MS QqQ

8

• ICP-MS QqQ utilizes tandem quadrupoles to isolate different elements which may have the same m/z increasing sensitivity for element of interest

• Examples include removal of 204Hg+

interference with 204Pb+ or trace detection of 129I

Ionization Source Q1 Q2Collision Cell Detector

Filter Desired Ion

Reactive Gas in

Collision Cell

Filter Desired Ion

Limit of Detection

9

• Analysis of a number of blanks will generate a distribution where Lc can be determined as a level above the blank

• Alpha represents the probability of a false positive• At Lc the chances of a false negative are 50%

https://www.intechopen.com/books/advances-in-gas-chromatography/limit-of-detection-and-limit-of-quantification-determination-in-gas-chromatography

Limit of Detection

10

• Limit of detection must be increased to a level where the risk of a false negative is reduced to an acceptable level

• LOD will be higher than Lc based on the standard deviation of the distribution and acceptable amount of error

• This is where S/N of 2-3 times noise arises

https://www.intechopen.com/books/advances-in-gas-chromatography/limit-of-detection-and-limit-of-quantification-determination-in-gas-chromatography

Signal-to-Noise

11

• Used for methods which produce back ground noise

• Baseline 20X the peak width at half height is analyzed

• S/N=2H/h• LOD=3:1• LOQ=10:1

h

H

S/N=2H/h

Blank Determination

12

• Used when blank gives non-zero standard deviation (noise)• Overlap of distributions of LOD and Blank can result in both false positives and false

negatives• LOD=Xb+3Sb

• LOQ=Xb+10Sb

Noise in Noiseless Detectors

13

• Decreased background can result in more varied noise regions

• Utilization of traditional signal-to-noise approach wont work for MRM acquisition as highly variable baseline noise results in highly variable LOD and LOQ determination

• Alternative methods for LOD and LOQ determination are necessary

Red-10 ppm in Scan

Blue-100 ppb in MRM

Baseline Noise in MRM

14

• In noiseless detectors selection of background noise region becomes extremely critical• Different locations can give highly variable amounts of background noise resulting in

inconsistent signal-to-noise ratios for the same analyte

Noise Region (a) (b) (c)

Time (min) 3-4 4.5-5.5 7.5-8.5

Signal to Noise 126 583 3594

Linear Regression

15

• Most applicable to methods where background noise is absent• LOD=3Sa/b• LOQ=10Sa/b• Standard deviation of the slope can be calculated from y-residuals

Instrument Detection Limit Testing Level

16

• Repeated analysis of a low concentration of analyte allows for statistical determination of LOD of instrument

• Critical that concentration analyzed is not too high or not too low

• Optimal %RSD 8-20%

7

9

11

13

15

17

19

0 10 20 30 40 50

% R

SD

Concentration (ppb)

Instrument Detection Limit

17

• Repeated analysis of a low concentration of analyte allows for statistical determination of LOD of instrument

• Critical that concentration analyzed is not too high or not too low

• Optimal %RSD 8-20%

7

9

11

13

15

17

19

0 10 20 30 40 50

% R

SD

Concentration (ppb)

Instrument Detection Limit

18

• Analysis of 7+ injections of the same concentration at predetermined level (8%-20% RSD) allows for instrument detection limit calculation to be performed

• IDL=t-value(99% CI)*RSD*Concentration/100

8 injections-99% CI-2.896

RSD-8.83%

𝐼𝐷𝐿 =2.896 ∗8.83%∗1 𝑝𝑝𝑏

100=256 ppt

Effect of Standard Deviation on IDL

19

• Standard deviation of analyzed concentration directly correlated to IDL• Smaller standard deviation given the same alpha and instrument sensitivity results

in a lower IDL

Mean Standard Response

Instrument Detection Limit

Quantity of Analyte

Sig

nal R

esponse

Mean Standard Response

Instrument Detection Limit

Quantity of Analyte

Sig

nal R

esponse

Method Detection Limit

20

• According to US EPA-The method detection limit (MDL) is defined as the minimum concentration of a substance that can be measured and reported with 99% confidence that the analyte concentration is greater than zero and is determined from analysis of a sample in a given matrix containing the analyte.

• Due to the possibility of matrix suppression or enhancement the method detection limit (MDL) is a more appropriate measure of LOD than using IDL

• Analyzing 7+ spiked samples, the MDL is calculated in the same manner as the IDL• Practical Quantitation Limit (PQL) is 5-10 times the MDL

Jordi Labs Analysis Using Noiseless Detection

21

• https://jordilabs.com/knb/case-studies/medical-grade-polymer/

• https://jordilabs.com/knb/case-studies/detecting-aflatoxins-medical-device-el/

In Summary

22

• No noise detectors require different methods to evaluate detection limits due to inconsistent or nonexistent baseline noise

• Statistical evaluation or linear regression approaches are most appropriate in noiseless detection modes

• USP, ICH, and FDA guidelines serve to inform determination of detection limit in noiseless detectors

23

Thank You!