1. James Byrd, Marta Kozak 28 Apr 2011
LC-MS analysis of six opiates in urine with Quantum Ultra triple quadrupole mass spectrometer
James Byrd, Marta Kozak
28 Apr 2011

2. Purpose Develop LC-MS method to analyze six opiates in urine
Method specification:
Analysis time: max 6 min
LOQ of 40 ng/mL (20 ng/mL even better) to support cut off value of 50 ng/mL
Sample preparation: glucuronide enzymatic hydrolysis followed by urine dilution

3. Conclusion
6 minutes method to analyzed six opiates in urine was developed
Method meets laboratory standards for linearity, accuracy and precision
Linearity: ng/mL
Precision:
QC1 (16.7 ng/mL): <15.7%
QC2 (167 ng/ml); <5.2
Accuracy:
QC1 (16.7 ng/ml): %
QC2 (167ng/ml): %
Method was tested for matrix effects-no significant matrix effects were observed

4. List of analytes and internal standards
Analayte
Internal Standard
Morphine
Morphine-D3
Hydromorphone
Hydromorphone-D6
Oxymorphone
Oxymorphone-D3
Codeine
Codeine-d3
Hydrocodone
Hydrocodone-D3
Oxycodone
Oxycodone-D3
Two pairs of analytes - Morphine-Hydromorphone and Codeine-Hydrocodone - are isobaric (have the same SRM transitions) and these compounds have to be chromatographically separated.

5. Method validation and acceptance criteria
Linearity range
Calibration standards acceptance criteria: 20% accuracy for the lowest calibrator and 15% accuracy for higher calibrators
Precision
%RSD equal to 20% or better for the low QC sample and equal to 15% or better for high QC sample analyzed in five replicates
Spike recovery
%Recovery within 30% for the low spiked concentration (10ng/mL) and within 20% for the high concentration (125 ng/mL)

6. Instrumentation
Quantum Ultra triple quadruple mass spectrometer with HESI source
TLX-1

7. Calibration standards
Calibration standards spiking solutions were prepared in MeOH by diluting 1 mg/mL solutions purchased from Cerillant
Calibration standards were prepared in urine by mixing 10 uL of standard spiking solution with 990 uL of blank urine

8. QC samples and spiked urine samples
Two QC samples with concentrations of 16.7 ng/mL and 167 ng/mL were prepared in urine
Six urines from different subjects were spiked with mix of analytes to final concentrations of 10 ng/mL and 125 ng/mL

9. Sample Processing Method
Aliquot 0.2 mL urine into microcentrifuge tube
Add 10 µL IS (4000 ng/mL of each deuterated analyte in MeOH)
Add 0.1 mL ß-glucuronidase (Sigma G-8132: dissolve at 10,000 U/mL in 1M acetate buffer adjusted to pH 5 w/glacial acetic acid,)
Incubate at 60°C for 60 min
Cool samples to room temperature
Add 200 µL MeOH
Vortex
Centrifuge at rpm for 5 minutes
Mix 0.05 mL supernatant mL water in HPLC vial
Inject µL into LC-MS system

10. LC method
Analytical Column: 50 x 4.3 mm, 1.9 um Hypersil GOLD aQ (part number )
Column temperature: 25 deg C
Mobile Phase
A: 10 mM NH4FA, 0.1%FA in DI water
B: 10 mM NH4FA, 0.1%FA in MeOH
C: ACN/IPA/Acetone=20/20/10/v/v/v
Gradient: total time = 5.67 min

11. Mass Spec source parameters
HESI source
Spray Voltage: 3500 mV
Heater temp: 350 deg C
Capillary temp: 370 deg C
Sheet gas: 45 units
Aux gas: 25 units
Probe position: C/D

12. Mass Spec acquisition method
Timed SRM transitions-two SRM transitions for each analyte
Cycle time 0.4 sec
Resolution: 0.7 FWHM
Collision gas pressure: 1.4 mTorr
Divert valve in 3 positions: minutes to mass spectrometer

13. Mass Spec acquisition method: List of SRM transitions and acquisition time windows

14. Mass Spec acquisition method-software screen capture

15. Mass spec acquisition method-divert valve positions

16. Processing method
Each analyte and internal standard were identified with ion ratio.
Ion ratio accuracy window was set to 30%(relative) for Morphine and Oxymorphone and to 20% (relative) for rest of analytes and internal standards
30%(relative) ion ratio accuracy windows for Morphine and Oxymorphone was needed to allow LOQ of 5 ng/mL for Morphine and 10 ng/mL for Oxymorphone.
If LOQ of 5-10 ng/mL is not required then ion ratio accuracy window can be set to 20% (relative)

17. Processing method: Morphine-D3 and Morphine

18. Results

19. Results: 10 ng/mL calibration standard in urine
Morphine
Morphine-D3
Hydromorphone
Hydromorphone-D6
Oxymorphone
Oxymorphone-D3
Codeine
Codeine-D3
Hydrocodone
Hydrocodone-D3
Oxycodone-D3
Oxycodone

20. Results Summary – Calibration curves: injection volume =20 uL
Morphine R2=0.9870
Hydromorphone R2=0.9874
Oxymorphone R2=0.9868
Cal Std
% Diff
10 ng/mL
-5.99
25 ng/mL
11.3
50 ng/mL
8.34
150 ng/mL
6.00
250 ng/mL
-10.9
500 ng/mL
-8.76
Cal Std
% Diff
10 ng/mL
-4.73
25 ng/mL
11.1
50 ng/mL
3.62
150 ng/mL
1.71
250 ng/mL
-16.4
500 ng/mL
4.64
Cal Std
% Diff
10 ng/mL
-1.05
25 ng/mL
-4.44
50 ng/mL
15.8
150 ng/mL
3.36
250 ng/mL
-13.8
500 ng/mL
0.19
Codeine R2=0.9967
Hydrocodone R2=0.9961
Oxycodone R2=0.9923
Cal Std
% Diff
10 ng/mL
2.24
25 ng/mL
-7.13
50 ng/mL
1.37
150 ng/mL
7.04
250 ng/mL
-2.84
500 ng/mL
-0.68
Cal Std
% Diff
10 ng/mL
-0.97
25 ng/mL
-0.24
50 ng/mL
7.53
150 ng/mL
-3.72
250 ng/mL
-7.00
500 ng/mL
4.40
Cal Std
% Diff
10 ng/mL
-1.65
25 ng/mL
-1.12
50 ng/mL
11.6
150 ng/mL
3.83
250 ng/mL
-10.8
500 ng/mL
-1.88

21. Internal standard signal precision in sequence of 19 samples
%RSD n=29
Morphine-D3
10.4
Hydromorphone-D6
10.5
Oxymorphone
Codeine
6.9
Hydrocodone
8.7
Oxycodone
5.3

22. Results Summary – QC1 samples: injection volume =20 uL
Spiked concentration = 16.7 ng/mL
Morphine %RSD=11.9
Hydromorphone %RSD=11.8
Oxymorphone %RSD=7.85
Sample #
Conc (ng/mL)
% Diff
QC1-1
15.5
-7.27
QC1-2
19.7
17.7
QC1-3
16.4
-1.56
QC1-4
20.7
23.8*
QC1-5
18.3
9.72
Sample #
Conc (ng/mL)
% Diff
QC1-1
18.3
9.78
QC1-2
21.6
29.2*
QC1-3
16.2
-3.0
QC1-4
20.0
19.7
QC1-5
17.0
1.78
Sample #
Conc (ng/mL)
% Diff
QC1-1
17.5
4.91
QC1-2
20.3
21.4*
QC1-3
17.3
3.60
QC1-4
20.4
21.9*
QC1-5
19.6
17.4
Codeine %RSD=11.4
Hydrocodone %RSD=15.7
Oxycodone %RSD=11.5
Sample #
Conc (ng/mL)
% Diff
QC1-1
17.8
6.78
QC1-2
20.0
20.1*
QC1-3
15.9
-4.81
QC1-4
19.6
17.4
QC1-5
15.7
-6.14
Sample #
Conc (ng/mL)
% Diff
QC1-1
17.5
4.89
QC1-2
22.1
32.4*
QC1-3
15.4
-7.91
QC1-4
19.2
15.0
QC1-5
15.5
-7.33
Sample #
Conc (ng/mL)
% Diff
QC1-1
15.8
-5.66
QC1-2
19.4
15.9
QC1-3
15.3
-8.25
QC1-4
17.8
6.79
QC1-5
14.8
-11.0
*) results which did not meet accuracy criteria of 20% if calculated against intended concentration of 16.7 ng/mL
All samples (except one) meet accuracy criteria if calculated against mean value

23. Results Summary – QC2 samples: injection volume =20 uL
Spiked concentration = 167 ng/mL
Morphine %RSD=5.06
Hydromorphone %RSD=5.16
Oxymorphone %RSD=4.06
Sample #
Conc (ng/mL)
% Diff
QC2-1
162
-3.13
QC2-2
165
-0.97
QC2-3
175
4.60
QC2-4
180
8.08
QC2-5
181
8.36
Sample #
Conc (ng/mL)
% Diff
QC2-1
154
-7.86
QC2-2
158
-5.42
QC2-3
168
0.34
QC2-4
174
4.09
QC2-5
170
2.12
Sample #
Conc (ng/mL)
% Diff
QC2-1
180
7.83
QC2-2
167
0.02
QC2-3
185
11.0
QC2-4
178
6.74
QC2-5
184
10.2
Codeine %RSD=1.75
Hydrocodone %RSD=4.10
Oxycodone %RSD=3.57
Sample #
Conc (ng/mL)
% Diff
QC2-1
171
2.32
QC2-2
170
1.65
QC2-3
174
4.18
QC2-4
168
0.92
QC2-5
176
5.29
Sample #
Conc (ng/mL)
% Diff
QC2-1
154
-7.48
QC2-2
-9.02
QC2-3
159
-4.66
QC2-4
166
-0.73
QC2-5
168
-0.74
Sample #
Conc (ng/mL)
% Diff
QC2-1
166
-0.75
QC2-2
155
-7.23
QC2-3
154
-7.52
QC2-4
160
-4.26
QC2-5
-0.31

24. Results Summary – Spiked urine Level 1: injection volume =20 uL
Spiked concentration = 10 ng/mL
Morphine
%RSD=13.7 Recovery %
Hydromorphone
%RSD= 19.7 Recovery = %
Oxymorphone
%RSD=7.0 Recovery= %
Cal Std
Conc (ng/mL)
% Diff
Urine#1
12.9
29.4
Urine#2
10.5
5.35
Urine#3
8.07
-19.3
Urine#4
7.35
Urine#5
24.3
Urine#6
46.4
Cal Std
Conc (ng/mL)
% Diff
Urine#1
8.76
-12.3
Urine#2
11.2
11.9
Urine#3
9.20
-7.95
Urine#4
11.1
10.6
Urine#5
12.0
20.5
Urine#6
12.3
23.5
Cal Std
Conc (ng/mL)
% Diff
Urine#1
10.2
2.18
Urine#2
11.9
18.8
Urine#3
11.0
9.72
Urine#4
10.3
2.64
Urine#5
12.0
19.5
Urine#6
10.7
6.89
Codeine
%RSD=14.0 Recovery= %
Hydrocodone
%RSD=9.7 Recovery %
Oxycodone
%RSD=13.2 Recovery=89-124%
Cal Std
Conc (ng/mL)
% Diff
Urine#1
10.4
3.88
Urine#2
7.65
-23.5
Urine#3
8.69
-13.1
Urine#4
8.98
-10.2
Urine#5
9.66
-3.39
Urine#6
11.4
14.2
Cal Std
Conc (ng/mL)
% Diff
Urine#1
12.7
26.7
Urine#2
11.5
15.0
Urine#3
9.89
-1.07
Urine#4
11.1
Urine#5
10.3
3.30
Urine#6
12.4
23.7
Cal Std
Conc (ng/mL)
% Diff
Urine#1
10.2
2.56
Urine#2
8.90
-11.0
Urine#3
9.44
-5.57
Urine#4
9.62
-3.76
Urine#5
11.7
17.0
Urine#6
12.4
23.9

25. Results Summary – Spiked urine Level 1: injection volume =20 uL
Spiked concentration = 10 ng/mL
Morphine
%RSD=13.7 Recovery = %
Hydromorphone
%RSD= 19.7 Recovery = %
Oxymorphone
%RSD=7.0 Recovery= %
Cal Std
Conc (ng/mL)
% Diff
Urine#1
12.9
29.4
Urine#2
10.5
5.35
Urine#3
8.07
-19.3
Urine#4
7.35
Urine#5
12.4
24.3
Urine#6
14.6
46.4
Cal Std
Conc (ng/mL)
% Diff
Urine#1
8.76
-12.3
Urine#2
11.2
11.9
Urine#3
9.20
-7.95
Urine#4
11.1
10.6
Urine#5
12.0
20.5
Urine#6
12.3
23.5
Cal Std
Conc (ng/mL)
% Diff
Urine#1
10.2
2.18
Urine#2
11.9
18.8
Urine#3
11.0
9.72
Urine#4
10.3
2.64
Urine#5
12.0
19.5
Urine#6
10.7
6.89
Codeine
%RSD=14.0 Recovery= %
Hydrocodone
%RSD=9.7 Recovery = %
Oxycodone
%RSD=13.2 Recovery= %
Cal Std
Conc (ng/mL)
% Diff
Urine#1
10.4
3.88
Urine#2
7.65
-23.5
Urine#3
8.69
-13.1
Urine#4
8.98
-10.2
Urine#5
9.66
-3.39
Urine#6
11.4
Cal Std
Conc (ng/mL)
% Diff
Urine#1
12.7
26.7
Urine#2
11.5
15.0
Urine#3
9.89
-1.07
Urine#4
11.1
Urine#5
10.3
3.30
Urine#6
12.4
23.7
Cal Std
Conc (ng/mL)
% Diff
Urine#1
10.2
2.56
Urine#2
8.90
-11.0
Urine#3
9.44
-5.57
Urine#4
9.62
-3.76
Urine#5
11.7
17.0
Urine#6
12.4
23.9

26. Results Summary – Spiked urine Level 2: injection volume =20 uL
Spiked concentration = 125 ng/mL
Morphine
%RSD=3.7 Recovery = %
Hydromorphone
%RSD= 8.4 Recovery = %
Oxymorphone
%RSD=7.57 Recovery= %
Cal Std
Conc (ng/mL)
% Diff
Urine#1
120
-4.16
Urine#2
131
5.20
Urine#3
125
0.322
Urine#4
119
-4.94
Urine#5
124
-0.978
Urine#6
-0.300
Cal Std
Conc (ng/mL)
% Diff
Urine#1
110
-11.9
Urine#2
119
-4.55
Urine#3
94.1
-24.7
Urine#4
115
-7.97
Urine#5
118
-5.28
Urine#6
116
-6.61
Cal Std
Conc (ng/mL)
% Diff
Urine#1
128
2.13
Urine#2
140
11.8
Urine#3
142
13.2
Urine#4
121
-3.08
Urine#5
135
8.68
Urine#6
150
20.0
Codeine
%RSD=6.8 Recovery= %
Hydrocodone
%RSD=11.1 Recovery %
Oxycodone
%RSD=6.0 Recovery= %
Cal Std
Conc (ng/mL)
% Diff
Urine#1
111
-11.2
Urine#2
125
-0.041
Urine#3
127
2.03
Urine#4
107
-14.6
Urine#5
119
-4.58
Urine#6
120
-4.04
Cal Std
Conc (ng/mL)
% Diff
Urine#1
97.0
-22.4
Urine#2
122
-2.61
Urine#3
109
-12.4
Urine#4
130
3.90
Urine#5
106
-14.8
Urine#6
102
-18.1
Cal Std
Conc (ng/mL)
% Diff
Urine#1
112
-10.2
Urine#2
123
-1.88
Urine#3
126
0.954
Urine#4
107
-14.4
Urine#5
119
-4.64
Urine#6
115
-7.34