Development and Validation of a Qualitative Method for Screening Pesticide Residues in Fruits and Vegetables using UHPLC/ESI Q-Orbitrap based on a Compound Database Approach
Jian Wang, PhDCalgary LaboratoryCanadian Food Inspection Agency
Outline• Objectives• Method development
• Data acquisition
• Build Compound Database (CDB)– Retention time correction
Outlin
e – Retention time correction– Set peak detection threshold
• Method validation
• Method performance parameters and criteria
• Results• Conclusion
2
Outlin
e
UHPLC/ESI Q-Orbitrap Applications for Chemical Contaminants Analysis
Quantification Target or Non-target Screening
1 2 3
1. Target compound analysis (Quantification)
2. Target compound screen (Compound Database or MS library)
3. Non-target compound screen (Differential Analysis)
Obje
ctiv
es
3
QuantificationScreening
Obje
ctiv
es
Rationale48th CCPR Proposed Draft Guidelines on Method Performance CriteriaThe screening concept offers laboratories an effective means to extend their analytical scope to analytes which potentially have a low probability of being present in the samples. Analytes that occur more frequently should continue to be monitored using validated quantitative multi-residue methods.
Obje
ctiv
es
4
DetectabilitySoftware detects the peaks of interest without manual integration1. Build Compound Database for
target screening2. Reduce false negative rate3. Reduce false positive rate
multi-residue methods.
Obje
ctiv
es
WORKFLOW
Meth
od D
evelo
pm
ent
5
Meth
od D
evelo
pm
ent
Mass [m/z] Polarity
167.07027 Positive
167.10397 Positive
168.06552 Positive
169.06816 Positive
170.09643 Positive
172.01640 Positive
174.06802 Positive
174.09134 Positive
179.12126 Positive
180.08012 Positive
180.08012 Positive
182.12879 Positive
183.11280 Positive
184.01918 Positive
184.07666 Positive
Inclusion list
184.07666 Positive
184.13052 Positive
185.03552 Positive
185.03552 Positive
185.07094 Positive
186.12370 Positive
187.08418 Positive
187.12298 Positive
188.06820 Positive
188.11036 Positive
189.08592 Positive
189.15975 Positive
190.04334 Positive
190.12601 Positive
191.08488 Positive
191.09457 Positive
6
Data
Acq
uis
itio
n Compound Database is built by injecting individual standards using Full MS/ddMS2 and “inclusion list” to obtain the product ion spectrum of a pesticide.
Sample data are
m/z 100 – 500m/z 25 × 16 = 400
m/z 500 – 900m/z 100 × 4 = 400
Sample data are acquired using Data Independent Acquisition (DIA) and, therefore, the data include all ions from HCD of a specific mass range.
7
Data
Acq
uis
itio
n
Identify the most intense precursor ion, normally in its protonated
Up to five ions come out from HCD are included in CDB from high to low according to ion intensity but the first one is always a precursor ion.
Build
Com
pound D
ata
base
its protonated form
Find individual fragments from ddMS2 product ion spectrum
1
2
Intensity high to low
Build
Com
pound D
ata
base
8
In spectral mass mass correction is based on precursor ion or 214.08963
Import from Excel to TraceFinder to build an executable Compound Database
Build
Com
pound D
ata
base
10
Build
Com
pound D
ata
base
Question: Why correct retention time in CDB?
Facts:1. Software chooses the nearest peak to the retention time of individual pesticides
specified in CDB.2. Software possibly picks up a wrong peak as a result of one or more adjacent
peaks close to the compound of interest.
Corr
ect
ion
RT uncorrected
11
Rete
ntion T
ime C
How to correct retention time?using 3-hydroxycarbofuran as a reference
Corr
ect
ion
Rete
ntion T
ime C
RT uncorrected
Retention time correction is critical to identify the compound of interest and to reduce false negative rate
Corr
ect
ion
RT corrected
Rete
ntion T
ime C
Fact:There are a significant number of false positives from a blank sample if data are processed by a default response threshold such as 30,000 for all pesticides.
Question: Why set peak detection threshold?ete
ctio
n T
hre
shold
14
Set
Peak D
ete
ctio
n
30,000
Response threshold can be set for individual pesticidesete
ctio
n T
hre
shold
15
Set
Peak D
ete
ctio
n
Each individual
How to set peak response
threshold?
Facts:1. Response (peak
area) is pesticide dependent.
2. Matrix effects lead to ion suppression or enhancement.
ete
ctio
n T
hre
shold
16
Each individual pesticide response threshold is calculated from the peak area of 5 ppb standard and is tested at different percentage of its original area counts.
OPA: original peak area
Set
Peak D
ete
ctio
n
10% original peak area@ 5 ppb
Setting response
threshold is
ete
ctio
n T
hre
shold
30,000 default response threshold
threshold is critical to
reduce false positive
rate.
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Set
Peak D
ete
ctio
n
Experimental design for method validation
Experiment design
� Matrix: 2 groups or 10 matrices including apple, banana, grape, orange, and strawberry, and carrot, potato, tomato, broccoli, lettuce.
� Spiked at 10 or 100 µg/kg in duplicate.
� Performed on 3 different days.
alid
ation
QuEChERs
18
Only 10 µg/kg, 1:1 dilution, results are presented here.
Meth
od V
alid
ation
QuEChERs� 15 g samples + 15 mL of acetonitrile/acetic
acid (99:1, v/v) + 1.5 g of sodium acetate anhydrous + 6.0 g of magnesium sulfate anhydrous
� 9 mL supernatants + 900 mg MgSO4, 150 mg C18 and 300 mg PSA
� Dilute 1:1 or concentrate 3:1 prior to UHPLC/ESI Q-Orbitrap
Screening method performance parameters and criteria• Parameters
• Retention Time and accurate mass of Precursor by Full MS Scan (RTP) for tentative positive screening.
• Retention Time and accurate mass of a Fragment Ion by DIA (RTFI) for confirmatory positive screening.
Para
mete
rs a
nd C
rite
ria
• Criteria• Retention time: ±0.2 or 0.5 min • Mass accuracy: ≤5 ppm
• An acceptable false-negative rate of ≤5%
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Meth
od P
erf
orm
ance
P
Resu
lts
ResultsAn acceptable false-negative rate of ≤ 5%
a Data by threshold of 30 K are based on ±0.2 min and Data by threshold of 10% are based on ±0.5 min sec.
Resu
lts
20
RTP Manual Check: to include or exclude any peaks missed or misidentified by software
The ideal scenario
An acceptable false-negative rate of ≤5%
Resu
lts
20 samples x 3 days x 450 = 27,000 data points
~50 pesticides can’t be detected due to low sensitivity or failing on the criterion of acceptable false-negative rate of ≤5%
21
Resu
lts
RTP Manual Check vs RTP (±0.2 min, ≤5 ppm)
An acceptable false-negative rate of ≤5%
Resu
lts
Tentative positive screening
22
Resu
lts
RTP Manual Check vs RTP vs FI vs RTFI (±0.2 min, ≤5 ppm)
An acceptable false-negative rate of ≤5%
Resu
lts
Confirmatory positive screening
23
Resu
lts
RTP: 30,000 default vs 10% threshold (±0.5 min, ≤5 ppm)
An acceptable false-negative rate of ≤5%
Resu
lts
Tentative positive screening
24
Resu
lts
Final screening method: RTP vs RTFI (±0.5 min, ≤5 ppm, 10% threshold)
An acceptable false-negative rate of ≤5%
Resu
lts
Tentative positive screening and Confirmatory positive screening
25
Resu
lts
Conclusion
UHPLC/ESI Q-Orbitrap along with Compound Database (CDB) can serve as a practical approach for pesticide residues screening in fruits and vegetables.� Retention time correction is critical to reduce false negative rate
and to detect true positives.
� Appropriate response threshold is critical to reduce false positive rate.
Concl
usi
on
rate.
� Tentative positive screening: Retention Time (±0.5 min ) and accurate mass (≤5 ppm) of Precursor by Full MS Scan (RTP).
� Detect ~400 (out of 450) pesticides at 10 µg/kg.� Confirmatory positive screening: Retention Time (±0.5 min ) and
accurate mass (≤5 ppm) of a Fragment Ion by DIA (RTFI).
� Detect ~330 (out of 450) pesticides at 10 µg/kg.
Concl
usi
on
26
Willis Chow, Canadian Food Inspection AgencyJames Chang, ThermoFisher Scientific Inc
Jon W Wong, US Food and Drug Administration
Acknowledgment
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