Clinical LC-MS/MS Systems: Analytical Capabilities · 2019. 4. 15. · 17-OHP 0.76–303 0.76 9.2% ≤8.2% ≤8.2% -5.0% DHEAS 140–54000 140 7.0% ≤3.9% ≤2.7% 5.8% Table 1. Performance

1

[ CLINICAL DIAGNOSTICS ]

Clinical LC-MS/MS Systems: Analytical Capabilities

2

[ ENDOCRINOLOGY ]

ACQUITY UPLC I-Class/Xevo TQD IVD System: Analytical Performance for Steroid Prohormones ......................................................................................................................3

ACQUITY UPLC I-Class/Xevo TQD IVD System: Analytical Performance for Progestogens and Androgens ....................................................................................................5

ACQUITY UPLC I-Class/Xevo TQD IVD System: Analytical Performance for Catecholamines and Metanephrines ....................................................................................... 7

ACQUITY UPLC I-Class/Xevo TQ-S micro IVD System: Analytical Performance for Progestogens and Androgens ....................................................................................................9

ACQUITY UPLC I-Class/Xevo TQ-S micro IVD System: Analytical Performance for Androgens, Progestogens and Glucocorticoids...................................................................................... 11

ACQUITY UPLC I-Class/Xevo TQ-S micro IVD System: Analytical Performance for a Mineralocorticoid ........................................................................................................................ 13

[ THERAPEUTIC DRUG MONITORNG ]

ACQUITY UPLC I-Class/Xevo TQD IVD System: Analytical Performance for an Inosine-5’-Monophosphate Dehydrogenase Inhibitor............................................................................................... 15

ACQUITY UPLC I-Class/Xevo TQD IVD System: Analytical Performance for an Alkylating Agent ........................................................................................................................ 17

ACQUITY UPLC I-Class/Xevo TQD IVD System: Analytical Performance for an Antifolate Agent ......................................................................................................................... 19

ACQUITY UPLC I-Class/Xevo TQD IVD System: Analytical Performance for Azole Antifungals ............................................................................................................................ 21

ACQUITY UPLC I-Class/Xevo TQD IVD System: Analytical Performance for a Thymidylate Synthase Inhibitor ........................................................................................... 23

ACQUITY UPLC I-Class/Xevo TQD IVD System: Analytical Performance for Immunosuppressive Agents ..................................................................................................... 25

Explore the analytical capabilities of Waters LC-MS/MS technology for analytes commonly used in the areas of endocrinology and therapeutic drug monitoring (TDM) work in the clinical laboratory.

3EndocrinologyReturn to Index

[ ANALYTICAL DATA BRIEF ]

EXPERIMENTAL DETAILSThe ACQUITY UPLC I-Class/Xevo TQD IVD System was controlled by MassLynx™ IVD Software (v4.1) and the data processed using the TargetLynx™ Application Manager. Calibrators and Quality Controls were prepared by spiking commercially available reference material in stripped serum and the samples were processed using the following conditions:

Sample preparation conditions150 µL sample was processed with zinc sulphate, methanol, and centrifuged. Samples were loaded onto Oasis™ HLB µElution plates, washed, and eluted prior to analysis.

LC conditionsColumn: ACQUITY UPLC BEH Phenyl (IVD)

1.7 µm, 2.1 mm × 50 mm

Pre-column: ACQUITY UPLC Column In-Line Filter

Mobile phase A: 2 mM Ammonium acetate + 0.1% formic acid in water

Mobile phase B: 2 mM Ammonium acetate + 0.1% formic acid in methanol

Flow rate: 0.45 mL/min

Gradient: 65–80% B over 2.5 minutes, 80% B over 0.2 minutes, 98% B for 0.8 minutes

MS conditionsResolution: MS1 (0.7 FWHM), MS2 (0.85 FWHM)

Acquisition mode: MRM

Polarity: ESI (+)

ACQUITY UPLC I-Class/Xevo TQD IVD System.

ACQUITY UPLC I-Class/Xevo TQD IVD System: Analytical Performance for Steroid Prohormones

INTRODUCTIONThe Waters™ ACQUITY™ UPLC™ I-Class/Xevo™ TQD IVD System enables the quantification of organic compounds in human biological liquid matrices.

This document describes a test of the analytical performance of the ACQUITY UPLC I-Class/Xevo TQD IVD System for the analysis of 25-hydroxyvitamin D2 (25OHD2) and 25-hydroxyvitamin D3 (25OHD3) in serum.

RESULTSPerformance characteristics of 25OHD2 and 25OHD3 are shown in Table 1. Chromatographic separation of 25OHD2 and 25OHD3 on the ACQUITY UPLC I-Class/Xevo TQD IVD System is illustrated in Figure 1.

4 Endocrinology Return to Index Return to Index

Waters Corporation 34 Maple Street Milford, MA 01757 U.S.A. T: 1 508 478 2000 F: 1 508 872 1990 www.waters.com


Waters, The Science of What’s Possible, ACQUITY, UPLC, MassLynx, Xevo, Oasis, and TargetLynx are trademarks of Waters Corporation. All other trademarks are the property of their respective owners.

©2018 Waters Corporation. Produced in the U.S.A. August 2018 720006356EN Rev. A AG-PDF

CompoundRange

(nmol/L)LLOQ (nmol)

Total precision

RepeatabilityDEQAS

mean bias25OHD2 10–375 3.0 ≤7.3% ≤6.5% N/A25OHD3 10–375 5.6 ≤6.6% ≤6.0% 2.1

Table 1. Performance characteristics of the analytes evaluated. Range defined by linear fit where r2>0.99. LLOQ defined by allowable precision and bias of 15% and 10% respectively, from samples performed over three days with two runs per day (n=90). Total Precision and Repeatability of samples performed over 20 occasions with two runs per day (n=80). Vitamin D External Quality Assessment Scheme (DEQAS) Mean Bias determined from NIST assigned DEQAS values.Note: To convert SI units to conventional mass units divide by 2.423 for 25OHD2 (nmol/L to ng/mL) and 2.496 for 25OHD3 (nmol/L to ng/mL).

Disclaimer The analytical performance data presented here is for illustrative purposes only. Waters does not recommend or suggest analysis of the analytes described herein. These data are intended solely to demonstrate the performance capabilities of the system for analytes representative of those commonly analyzed using liquid chromatography and tandem mass spectrometry. Performance in an individual laboratory may differ due to a number of factors, including laboratory methods, materials used, intra-operator technique, and system conditions. This document does not constitute a warranty of merchantability or fitness for any particular purpose, express or implied, including for the testing of the analytes in this analysis.

CONCLUSIONSThe Waters ACQUITY UPLC I-Class/Xevo TQD IVD System has demonstrated the capability to quantify 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 in serum with precision and accuracy.

For in vitro diagnostic use. Not available in all countries.

25OHD3 Qualifier Ion

min1.60 1.70 1.80 1.90 2.00 2.10 2.20 2.30 2.40 2.50 2.60 2.70 2.80 2.90 3.00 3.10 3.20 3.30 3.40

%

0

100

min

%

0

100

min

%

100

0

min1.60 1.70 1.80 1.90 2.00 2.10 2.20 2.30 2.40 2.50 2.60 2.70 2.80 2.90 3.00 3.10 3.20 3.30 3.40

%

0

100

min

%

0

100

min

%

0

10025OHD2 SampleNominal Conc. = 22.3 nmol/L (9.2 ng/mL)

Quantifier Ion (m/z 413.3 > 355.3)

25OHD3 SampleNominal Conc. = 20.3 nmol/L (8.1 ng/mL)

Quantifier Ion (m/z 401.3 > 159.1)

Qualifier Ion(m/z 413.3 > 83.1)

Qualifier Ion (m/z 401.3 > 365.3)

[2H3]-25OHD3 Internal Standard(m/z 404.3 > 162.1)

[2H3]-25OHD2 Internal Standard(m/z 416.3 > 358.3)

Figure 1. Chromatographic separation of 25OHD2 and 25OHD3 in a low level sample using the ACQUITY UPLC I-Class/Xevo TQD IVD System.

5EndocrinologyReturn to Index Return to Index


EXPERIMENTAL DETAILSThe ACQUITY UPLC I-Class/Xevo TQD IVD System was controlled by MassLynx™ IVD Software (v4.1) and the data processed using the TargetLynx™ Application Manager. Calibrators and Quality Controls were prepared by spiking commercially available reference material in stripped serum and the samples were processed using the following conditions:

Sample preparation conditions100 µL sample was precipitated with methanol, diluted with water, and centrifuged. Samples were loaded onto Oasis™ PRiME HLB µElution plates, washed, and eluted prior to analysis.

LC conditionsColumn: ACQUITY UPLC HSS T3 (IVD)

1.8 µm, 2.1 mm × 50 mm

Pre-column: VanGuard™ HSS T3 1.8 µm, 2.1 mm × 5 mm




Gradient: 45% B over one minute, 45–65% B over 2.5 minutes, 98% B for 0.5 minutes



Polarity: ESI (+/-)


ACQUITY UPLC I-Class/Xevo TQD IVD System: Analytical Performance for Progestogens and Androgens


This document describes a test of the analytical performance of the ACQUITY UPLC I-Class/Xevo TQD IVD System for the analysis of testosterone, androstenedione, 17-hydroxyprogesterone (17-OHP), and dehydroepiandrosterone sulfate (DHEAS) in serum.

RESULTSPerformance characteristics of the steroid hormones on the Waters ACQUITY UPLC I-Class/Xevo TQD IVD System are shown in Table 1. Analytical sensitivity of the system for analyzing the steroid hormones in plasma is illustrated in Figure 1.




Waters, The Science of What’s Possible, ACQUITY, UPLC, VanGuard, MassLynx, Xevo, Oasis, and TargetLynx are trademarks of Waters Corporation. All other trademarks are the property of their respective owners.


Figure 1. Low steroid hormone concentrations in serum containing DHEAS, androstenedione, testosterone, and 17-OHP.

Time2.00 2.50 3.00 3.50

%

0

100

2.00 2.50 3.00 3.50

%

0

100

2.00 2.50 3.00 3.50

%

0

100

2.00 2.50 3.00 3.50

%

0

100

Androstenedione

Testosterone

17-OHP

DHEAS

m/z 287.2>97.0

m/z 289.2>97.0

m/z 331.2>97.0

m/z 367.2>97.0

1.26 nmol/LS/N: PtP = 80



2520 nmol/LS/N: PtP = 2695

CompoundRange

(nmol/L)LLOQ (nmol)

%RSD at LLOQ

Total precision

RepeatabilityEQA

mean biasTestosterone 0.17–69 0.17 12.0% ≤4.7% ≤3.6% -0.5%

Androstenedione 0.17–69 0.17 9.1% ≤6.3% ≤5.2% 0.4%17-OHP 0.76–303 0.76 9.2% ≤8.2% ≤8.2% -5.0%DHEAS 140–54000 140 7.0% ≤3.9% ≤2.7% 5.8%

Table 1. Performance characteristics of the analytes evaluated. Range defined by linear fit where r 2 >0.99. LLOQ defined by S/N (PtP) >10 and %RSD <20%. % RSD at LLOQ determined through analytical sensitivity experiments performed over three occasions (n=30). Total Precision and Repeatability of QCs performed over five occasions in stripped serum (n=30). EQA Mean Bias determined through Altman-Bland comparison of calculated concentrations to EQA mass spectrometry mean values.Note: To convert SI units to conventional mass units divide by 3.470 for testosterone (nmol/L to ng/mL), 3.494 for androstenedione (nmol/L to ng/mL), 3.028 for 17-OHP (nmol/L to ng/mL) and 2.716 for DHEAS (nmol/L to ng/mL).


CONCLUSIONSThe Waters ACQUITY UPLC I-Class/Xevo TQD IVD System has demonstrated the capability to deliver analytically sensitive, selective performance with excellent precision and accuracy for testosterone, androstenedione, 17-OHP, and DHEAS in serum.




EXPERIMENTAL DETAILSThe ACQUITY UPLC I-Class/Xevo TQD IVD System was controlled by MassLynx™ IVD Software (v4.1) and the data processed using the TargetLynx™ Application Manager. Calibrators and Quality Controls were prepared by spiking commercially available reference material in urine and the samples were processed using the following conditions:

Sample preparation conditions400 µL acidified urine was diluted with 1 mL of 0.5 M ammonium acetate buffer. Samples were loaded onto Oasis™ WCX 30 mg plates, washed, and eluted prior to analysis.

LC conditionsColumn: ACQUITY UPLC BEH Amide

1.7 µm, 2.1 mm × 100 mm

Mobile phase A: 95:5 Water:acetonitrile containing 50 mM NH4HCOO, pH 3.0

Mobile phase B: 15:85 Water:acetonitrile containing 30 mM NH4HCOO, pH 3.0


Gradient: 100% B over 1 minute, 100–90% B from 1–2 minutes, 90% B at 1.0 mL/min at 2.1 minutes, 90–70% B from 2.1–2.5 minutes



Polarity: ESI (+)

ACQUITY UPLC I-Class/Xevo TQD IVD System: Analytical Performance for Catecholamines and Metanephrines

INTRODUCTIONThe Waters ACQUITY™ UPLC™ I-Class/Xevo™ TQD IVD System enables the quantification of organic compounds in human biological liquid matrices.

This document describes a test of the analytical performance of the ACQUITY UPLC I-Class/Xevo TQD IVD System for the analysis of norepinephrine, epinephrine, dopamine, normetanephrine, metanephrine, and 3-methoxytyramine in urine.

RESULTSPerformance characteristics of catecholamines and metanephrines on the ACQUITY UPLC I-Class/Xevo TQD IVD System are shown in Table 1. Analytical selectivity of the chromatographic separation is illustrated in Figure 1.

ACQUITY UPLCI-Class/Xevo TQD IVD System.




Waters, The Science of What’s Possible, ACQUITY, UPLC, MassLynx, Xevo, Oasis, and TargetLynx are trademarks of Waters Corporation. All other trademarks are the property of their respective owners.

©2018 Waters Corporation. Produced in the U.S.A. July 2018 720006341EN AG-PDF

Figure 1. Chromatographic selectivity of catecholamines and metanephrines using the ACQUITY UPLC I-Class/Xevo TQD IVD System. Note: 3-MT – 3-methoxytyramine; MTN – metanephrine; DA – dopamine; NMT – normetanephrine; EP – epinephrine; NE – norepinephrine.

Time0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40

%

0

100

0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40

%

0

100

0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40

%

0

100

0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40

%

0

100

0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40

%

0

1000.83

0.89

1.22

1.16

1.38

1.92

3-MT

MTN

DA

EPNMT

NE

m/z 168.1>91

m/z 198.1>180

m/z 154.0>91

m/z 184.1>166

m/z 170>152

CompoundRange

(ng/mL)LLOQ

(ng/mL)%RSD

at LLOQMax

imprecisionMax bias

3-methoxytyramine 21.7–521.2 21.7 3.1% 5.0% 9.8%Metanephrine 11.2–510.7 10.7 1.8% 2.9% 4.0%

Normetanephrine 18.3–517.8 17.8 1.1% 4.2% 3.4%Epinephrine 0.5–500 0.5 8.6% 6.2% -4.6%Dopamine 6.5–506 6.0 4.2% 11% -7.9%

Norepinephrine 5.1–504.6 4.6 16.3% 14.8% -6.0%



CONCLUSIONSThe Waters™ ACQUITY UPLC I-Class/Xevo TQD IVD System has demonstrated the capability to deliver analytically sensitive and precise chromatography for the analysis of 3-methoxytyramine, metanephrine, normetanephrine, dopamine, epinephrine, and norepinephrine in urine.

Table 1. Performance characteristics of the analytes evaluated. Range defined by linear fit where r2>0.99. LLOQ defined by S/N (PtP) >10 and %RSD ≤20%. %RSD at LLOQ determined through analytical sensitivity experiments (n=5). Maximum imprecision and bias determined over four concentrations (N=4). Note: To convert conventional mass units to SI units multiply by 5.98 for 3-MT, 5.07 for MTN, 5.46 for NMT and EP, 6.53 for DA, and 5.91 for NE. All conversions are from ng/mL to nmol/L.



EXPERIMENTAL DETAILSThe ACQUITY UPLC I-Class/Xevo TQ-S micro IVD System was controlled by MassLynx™ IVD Software (v4.1) and the data processed using the TargetLynx™ Application Manager. Calibrators and Quality Controls were prepared by spiking commercially available reference material in stripped serum and the samples were processed using the following conditions:

Sample preparation conditions100 µL sample was processed with methanol, diluted with water, and centrifuged. Samples were loaded on Oasis™ MAX µElution plates, washed, and eluted prior to analysis.

LC conditionsColumn: CORTECS UPLC C18

1.6 µm, 2.1 mm × 50 mm

Mobile phase A: 0.05 mM Ammonium fluoride in water

Mobile phase B: Methanol

Flow Rate: 0.25 mL/min

Gradient: 40% B over 0.5 minutes, 40–70% B over 3.5 minutes, 95% B for 0.5 minutes



Polarity: ESI (+)

ACQUITY UPLC I-Class/Xevo TQ-S micro IVD System.

ACQUITY UPLC I-Class/Xevo TQ-S micro IVD System: Analytical Performance for Progestogens and Androgens

INTRODUCTIONThe Waters™ ACQUITY™ UPLC™ I-Class/Xevo™ TQ-S micro IVD System enables the quantification of organic compounds in human biological liquid matrices.

This document describes a test of the analytical performance of the ACQUITY I-Class/Xevo TQ-S micro IVD System for the analysis of dihydrotestosterone (DHT), dehydroepiandrosterone (DHEA), testosterone, androstenedione, 17-hydroxyprogesterone, and progesterone in serum.

RESULTSPerformance characteristics of the steroid hormones on the ACQUITY UPLC I-Class/Xevo TQ-S micro IVD System are shown in Table 1. Analytical sensitivity of the system for analysing extracted steroid hormone samples is illustrated in Figure 1.




Waters, The Science of What’s Possible, ACQUITY, UPLC, MassLynx, Xevo, Oasis, CORTECS, and TargetLynx are trademarks of Waters Corporation. All other trademarks are the property of their respective owners.


Figure 1. Performance characteristics of low concentration serum steroid hormone samples using the ACQUITY UPLC I-Class/ Xevo TQ-S micro IVD System.

Androstenedione

Testosterone

17OHP

DHEA

DHT

Progesterone

m/z 287.2>97.1

m/z 289.2>97.1

m/z 331.2>97.1

m/z 271.2>213.2

m/z 315.2>97.1

1.17 nmol/L

S/N (PtP) = 330

0.77 nmol/L

S/N (PtP) = 717

1.09 nmol/L

S/N ( PtP) = 206

1.14 nmol/L

S/N (PtP) = 43

0.21 nmol/L

S/N ( PtP) = 38

0.09 nmol/L

S/N (PtP) = 15

Time 3.25 3.50 3.75 4.00 4.25 4.50 4.75 5.00

%

0

100

3.25 3.50 3.75 4.00 4.25 4.50 4.75 5.00

%

0

100

3.25 3.50 3.75 4.00 4.25 4.50 4.75 5.00

%

0

100

3.25 3.50 3.75 4.00 4.25 4.50 4.75 5.00

%

0

100

3.25 3.50 3.75 4.00 4.25 4.50 4.75 5.00

%

0

100

3.25 3.50 3.75 4.00 4.25 4.50 4.75 5.00

%

0

100

m/z 291.2>255.2

CompoundRange

(nmol/L)LLOQ

(nmol/L)%RSD at

LLOQTotal

precisionRepeatability Mean bias

DHT 0.086–34 0.086 13% ≤6.5% ≤6.4% 4.9%DHEA 1.0–69 0.35 11% ≤4.7% ≤4.4% –

Testosterone 0.017–69 0.017 11% ≤5.3% ≤2.9% -1.4%Androstenedione 0.087–349 0.035 18% ≤5.4% ≤3.7% 0.2%

17-OHP 0.076–303 0.030 17% ≤4.4% ≤4.0% -5.6%Progesterone 0.064–64 0.016 15% ≤4.5% ≤4.1% –

Table 1. Performance characteristics of the analytes evaluated. Range defined by linear fit where r2 >0.99. LLOQ defined by S/N (PtP) >10 and %RSD ≤20%. %RSD at LLOQ determined through analytical sensitivity experiments performed over five occasions (n=40). Total precision and repeatability of QCs performed over five occasions (n=25). Mean bias determined through Bland-Altman comparison of calculated concentrations to RCPA QAP target values for DHT and EQA mass spectrometry mean values for testosterone, androstenedione, and 17-OHP.Note: To convert SI units to conventional mass units divide by 3.45 for DHT (nmol/L to ng/mL), 3.47 for DHEA (nmol/L to ng/mL), 3.47 for testosterone (nmol/L to ng/mL), 3.49 for androstenedione (nmol/L to ng/mL), 3.03 for 17-OHP (nmol/L to ng/mL), and 3.18 for progesterone (nmol/L to ng/mL).



CONCLUSIONSThe ACQUITY UPLC I-Class/Xevo TQ-S micro IVD System has demonstrated the capability to deliver analytically sensitive and selective performance with excellent precision and accuracy for DHT, DHEA, testosterone, androstenedione, 17-hydroxyprogesterone, and progesterone in serum.



ACQUITY UPLC I-Class/ Xevo TQ-S micro IVD System.


Sample preparation conditions100 µL sample was precipitated with methanol, diluted with water, and centrifuged. Samples were loaded on Oasis™ PRiME HLB µElution plates, washed, and eluted prior to analysis.


1.8 µm, 2.1 × 50 mm

Pre-column: VanGuard™ HSS T3 1.8 µm, 2.1 × 5 mm

Mobile phase A: 2 mM Ammonium acetate +0.1% formic acid in water

Mobile phase B: 2 mM Ammonium acetate +0.1% formic acid in methanol


Gradient: 45% B over one minute, 45–65% B over 2.5 minutes, 98% B for 0.5 minutes



Polarity: ESI (+/-) Figure 1. Chromatographic selectivity of a range of steroids using the ACQUITY UPLC I-Class/Xevo TQ-S micro IVD System.

ACQUITY UPLC I-Class/Xevo TQ-S micro IVD System: Analytical Performance for Androgens, Progestogens and Glucocorticoids

INTRODUCTIONThe Waters ACQUITY™ UPLC™ I-Class/Xevo™ TQ-S micro IVD System enables the quantification of organic compounds in human biological liquid matrices.

This document describes a test of the analytical performance of the ACQUITY UPLC I-Class/Xevo TQ-S micro IVD System for the analysis of testosterone, androstenedione, 17-hydroxyprogesterone, dehydroepiandrosterone sulfate, cortisol, 11-deoxycortisol, and 21-deoxycortisol in serum.

RESULTSAnalytical selectivity of the chromatographic separation is illustrated in Figure 1. Performance characteristics of the steroid hormones on the ACQUITY UPLC I-Class/Xevo TQ-S micro IVD System are shown in Table 1.




Waters, The Science of What’s Possible, ACQUITY, UPLC, MassLynx, Xevo, Oasis, VanGuard, and TargetLynx are trademarks of Waters Corporation. All other trademarks are the property of their respective owners.


CONCLUSIONSThe Waters ACQUITY UPLC I-Class/Xevo TQ-S micro IVD System has demonstrated the capability to deliver analytically sensitive, selective performance with excellent precision and accuracy for testosterone, androstenedione, 17-hydroxyprogesterone, dehydroepiandrosterone sulfate, cortisol, 11-deoxycortisol, and 21-deoxycortisol in serum.


CompoundRange

(nmol/L)LLOQ (nmol)

%RSD at LLOQ

Total precision

RepeatabilityEQA

mean biasTestosterone 0.1–69 0.10 4.9% ≤6.3% ≤3.2% -0.1%

Androstenedione 0.09–349 0.09 9.1% ≤6.0% ≤4.0% -5.1%17-OHP 0.19–757 0.19 8.2% ≤5.3% ≤3.4% 5.2%DHEAS 65–43000 65 4.0% ≤7.6% ≤3.9% -5.8%Cortisol 0.69–1380 0.69 13.2% ≤7.3% ≤7.3% 1.0%

11-Deoxycortisol 0.72–144 0.72 14.1% ≤5.7% ≤3.5% –21-Deoxycortisol 0.72–144 0.72 9.9% ≤6.9% ≤5.2% –

Table 1. Performance characteristics of the analytes evaluated. Range defined by linear fit where r2 >0.99. LLOQ defined by S/N (PtP) >10 and %RSD <20%. %RSD at LLOQ determined through analytical sensitivity experiments performed over three occasions (n=30). Total precision and repeatability of QCs performed over five occasions in stripped serum (n=25). EQA mean bias determined through Altman-Bland comparison of calculated concentrations to EQA mass spectrometry mean values.Note: To convert SI units to conventional mass units divide by 3.470 for testosterone (nmol/L to ng/mL), 3.494 for androstenedione (nmol/L to ng/mL), 3.028 for 17-OHP (nmol/L to ng/mL), 2.716 for DHEAS (nmol/L to ng/mL), 2.761 for cortisol (nmol/L to ng/mL), and 2.889 for 11-deoxycortisol and 21-deoxycortisol (nmol/L to ng/mL).





Sample preparation conditions200 µL sample was processed with ZnSO4/methanol, diluted, and centrifuged. Samples were loaded onto Oasis™ MAX µElution plates, washed, and eluted prior to analysis.

LC conditionsColumn: CORTECS™ UPLC C18

1.6 µm, 2.1 mm × 100 mm

Mobile phase A: Water

Mobile phase B: Methanol


Gradient: 40% B over one minute, 40–60% B over one minute, 60% for 0.3 minutes, 95% B for 0.5 minutes



Polarity: ESI (-)

ACQUITY UPLC I-Class/Xevo TQ-S micro IVD System.

ACQUITY UPLC I-Class/Xevo TQ-S micro IVD System: Analytical Performance for a Mineralocorticoid

INTRODUCTIONThe Waters ACQUITY™ UPLC™ I-Class/Xevo™ TQ-S micro IVD System enables the quantification of organic compounds in human biological liquid matrices.

This document describes a test of the analytical performance of the ACQUITY UPLC I-Class/Xevo TQ-S micro IVD System for the analysis of aldosterone in plasma.

RESULTSPerformance characteristics of aldosterone using the ACQUITY UPLC I-Class/Xevo TQ-S micro IVD System is shown in Table 1. Analytical sensitivity of the system for analyzing extracted aldosterone plasma samples is illustrated in Figure 1.

Compound Range (pmol/L)

LLOQ (pmol/L)

S/N at LLOQ

Total precision Repeatability

Aldosterone 42–4161 42 37 ≤7.2% ≤7.0% Table 1. Performance characteristics of aldosterone. Range defined by linear fit where r2 >0.99. LLOQ defined by S/N (PtP) >10 and %RSD ≤20%. S/N at LLOQ determined using the mean S/N (PtP) at the low calibrator over five occasions. Total precision and repeatability of QCs performed over five occasions in plasma (n=25). Note: To convert SI units to conventional mass units divide by 2.774 for aldosterone (pmol/L to pg/mL).

Return to Index Return to Index14 Endocrinology Return to Index Return to Index



Waters, The Science of What’s Possible, ACQUITY, UPLC, MassLynx, Xevo, Oasis, CORTECS, and TargetLynx are trademarks of Waters Corporation. All other trademarks are the property of their respective owners.


Figure 1. Performance characteristics of extracted plasma aldosterone samples using the ACQUITY UPLC I-Class/Xevo TQ-S micro IVD System.

m/z 359.2>189.2

m/z 359.2>189.2

m/z 359.2>189.2

m/z 359.2>189.2

m/z 359.2>189.2

49 pmol /LS/N:PtP = 20





Aldosterone

Aldosterone

Aldosterone

Aldosterone

Aldosterone

Time1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00

%

0

100

1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00

%

0

100

1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00

%

0

100

1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00

%

0

100

1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00

%

0

100


CONCLUSIONSThe Waters ACQUITY UPLC I-Class/Xevo TQ-S micro IVD System has demonstrated the capability to deliver analytical sensitivity and precision for the analysis of aldosterone in plasma.


15Therapeutic Drug MonitorngReturn to Index Return to IndexReturn to Index Return to Index


EXPERIMENTAL DETAILSThe ACQUITY UPLC I-Class/Xevo TQD IVD System was controlled by MassLynx™ IVD Software (v4.1) and the data processed using the TargetLynx™ Application Manager. Commercial calibrators and Quality Controls were used as well as in-house calibrators prepared by spiking commercially available reference material in plasma. The samples were processed using the following conditions:

Sample preparation conditions50 µL sample was processed with zinc sulphate/methanol and centrifuged. The supernatants were transferred to a collection plate for analysis.

LC conditionsColumn: ACQUITY UPLC HSS C18 SB

1.8 µm, 2.1 mm × 30 mm




Gradient: 30–40% B over 0.75 minutes, 40–75% B over 0.85 minutes, 98% B for 0.4 minutes



Polarity: ESI (+)

ACQUITY UPLC I-Class/Xevo TQD IVD System: Analytical Performance for an Inosine-5'-Monophosphate Dehydrogenase Inhibitor


This document describes a test of the analytical performance of the ACQUITY UPLC I-Class/Xevo TQD IVD System for the analysis of mycophenolic acid in plasma.

RESULTSPerformance characteristics of mycophenolic acid on the ACQUITY UPLC I-Class/Xevo TQD IVD System are shown in Table 1. A chromatogram illustrating the selectivity of the mycophenolic acid analysis is shown in Figure 1.


16 Therapeutic Drug Monitorng Return to Index Return to Index



Waters, The Science of What’s Possible, ACQUITY, UPLC, MassLynx, Xevo, and TargetLynx are trademarks of Waters Corporation. All other trademarks are the property of their respective owners.


Figure 1. Chromatographic separation of mycophenolic acid from its metabolites using the ACQUITY UPLC I-Class/Xevo TQD IVD System.

Mycophenolic acid (MPA)

321.1>207.1

Mycophenolic acid acyl-glucuronide (AcMPAG)

Mycophenolic acid glucuronide (MPAG)

CompoundRange

(µg/mL)LLOQ

(µg/mL)%RSD

at LLOQTotal

precisionRepeatability

EQA mean bias

Mycophenolic acid 0.1–20 0.075 5.6% ≤5.3% ≤3.6% ≤5.1% Table 1. Performance characteristics of mycophenolic acid. Range defined by linear fit where r2 >0.99. LLOQ defined by S/N (PtP) >10 and %RSD ≤20%. %RSD at LLOQ determined through analytical sensitivity experiments performed over three occasions (n=30). Total precision and repeatability of QCs performed over five occasions in plasma (n=25). EQA mean bias determined by comparison of obtained values to HPLC-MS method mean.



CONCLUSIONThe Waters ACQUITY UPLC I-Class/Xevo TQD IVD System has demonstrated the capability to deliver analytically sensitive, accurate, and precise performance for mycophenolic acid in plasma.

17Therapeutic Drug MonitorngReturn to Index Return to Index


EXPERIMENTAL DETAILSThe ACQUITY UPLC I-Class/Xevo TQD IVD system was controlled by MassLynx™ IVD (v4.1) and the data processed using the TargetLynx™ Application Manager. Calibrators and Quality Controls were prepared by spiking commercially available reference material in plasma and the samples were processed using the following conditions:

Sample preparation conditions50 µL sample was processed with methanol and centrifuged, then subsequently diluted with water prior to analysis.


1.8 µm, 2.1 mm × 50 mm

Mobile phase A: 2 mM Ammonium acetate +0.1% formic acid in water

Mobile phase B: 2 mM Ammonium acetate +0.1% formic acid in methanol


Gradient: 10% B for 0.5 minutes, 10–98% B over 1.0 minute, 98% B for 0.5 minutes, 10% B for 0.5 minutes



Polarity: ESI (+)


ACQUITY UPLC I-Class/Xevo TQD IVD System: Analytical Performance for an Alkylating Agent


This document describes a test of the analytical performance of the ACQUITY UPLC I-Class/Xevo TQD IVD System for the analysis of busulfan in plasma.

RESULTSPerformance characteristics of busulfan on the ACQUITY UPLC I-Class/Xevo TQD IVD System are shown in Table 1. A chromatogram illustrating the analytical sensitivity of the busulfan analysis is shown in Figure 1.

Compound Range (µg/mL)

LLOQ (µg/mL)

%RSD at

LLOQ


Busulfan 0.025–5 0.02 16.0 ≤7.3% ≤5.1% Table 1. Performance characteristics of busulfan. Range defined by linear fit where r 2 >0.99. LLOQ defined by S/N (PtP) >10 and %RSD ≤20%. %RSD at LLOQ determined through analytical sensitivity experiments performed over five occasions (n=50). Total precision and repeatability of QCs performed over five occasions in plasma (n=25).




Waters, The Science of What’s Possible, ACQUITY, UPLC, and Xevo are trademarks of Waters Corporation. All other trademarks are the property of their respective owners.


Figure 1. Chromatogram showing the analysis of busulfan using the ACQUITY UPLC I-Class/Xevo TQD IVD System.

Busulfan- 2H8 ISTD m/z 272.0 -> 159.1

Busulfan Quantifier m/z 264.0 -> 55.0

Busulfan Quantifier m/z 264.0 -> 151.1


CONCLUSIONSThe Waters ACQUITY UPLC I-Class/Xevo TQD IVD System has demonstrated the capability to deliver analytical sensitivity and precision for the analysis of busulfan in plasma.




EXPERIMENTAL DETAILSThe ACQUITY UPLC I-Class/Xevo TQD IVD System was controlled by MassLynx™ IVD Software (v4.1) and the data processed using the TargetLynx™ Application Manager. Calibrators and Quality Controls were prepared by spiking commercially available reference material in plasma. The samples were processed using the following conditions:


LC conditionsColumn: ACQUITY UPLC HSS C18 SB

1.8 µm, 2.1 mm × 30 mm




Gradient: 77% A isocratic for 5 min



Polarity: ESI (+)


ACQUITY UPLC I-Class/Xevo TQD IVD System: Analytical Performance for an Antifolate Agent


This document describes a test of the analytical performance of the ACQUITY UPLC I-Class/Xevo TQD IVD System for the analysis of methotrexate in plasma.

RESULTSPerformance characteristics of methotrexate on the ACQUITY UPLC I-Class/Xevo TQD IVD System are shown in Table 1. Analytical sensitivity of the chromatographic separation is illustrated in Figure 1.






Figure 1. Chromatogram showing the analysis of methotrexate using the ACQUITY UPLC I-Class/Xevo TQD IVD System.

Methotrexate Quantifierm/z 455.2 -> 175.1

Methotrexate Qualifierm/z 455.2 -> 134.1

Methotrexate 2H3 Internal Standardm/z 458.2 -> 175.1

CompoundRange

(µmol/L)LLOQ

(µmol/L)%RSD at

LLOQTotal precision Repeatability EQA mean bias

Methotrexate 0.025–10 0.0025 15.7 ≤5.5% ≤4.0% -5.7% Table 1. Performance characteristics of methotrexate. Range defined by linear fit where r2 >0.99. LLOQ defined by S/N (PtP) >10 and %RSD ≤20%. %RSD at LLOQ determined through analytical sensitivity experiments performed over three occasions (n=30). Total precision and repeatability of QCs performed over five occasions in plasma (n=25). EQA mean bias determined by comparison of obtained values to the gravimetric reference value.Note: To convert SI units to conventional mass units divide by 2.2005 (µmol/L to µg/mL).

CONCLUSIONSThe Waters ACQUITY UPLC I-Class/Xevo TQD IVD System has demonstrated the capability to deliver an analytically sensitive and precise method for methotrexate in plasma.





EXPERIMENTAL DETAILSThe ACQUITY UPLC I-Class/Xevo TQD IVD System was controlled by MassLynx™ IVD Software (v4.1) and the data processed using the TargetLynx™ Application Manager. Calibrators and Quality Controls were prepared by spiking commercially available reference material in serum and the samples were processed using the following conditions:


LC conditionsColumn: ACQUITY UPLC BEH C18

1.7 µm, 2.1 mm × 30 mm




Gradient: 75% A initial, gradient 7 until 97% B at 2.1 minutes, then hold 75% A until 2.5 minutes



Polarity: ESI (+)


ACQUITY UPLC I-Class/Xevo TQD IVD System: Analytical Performance for Azole Antifungals


This document describes a test of the analytical performance of the ACQUITY UPLC I-Class/Xevo TQD IVD System for the analysis of fluconazole, hydroxyitraconazole, itraconazole, posaconazole, voriconazole, and voriconazole-N-oxide in serum.

RESULTSPerformance characteristics of the azole antifungals on the ACQUITY UPLC I-Class/Xevo TQD IVD System are shown in Table 1. Analytical selectivity of the chromatographic separation is illustrated in Figure 1.





©2018 Waters Corporation. Produced in the U.S.A. June 2018 720006319EN AG-PDF

Figure 1. Chromatographic separation of a range of azole antifungals using the ACQUITY UPLC I-Class/Xevo TQD IVD System.

Fluconazolem/z 307.1 -> 238.1

Voriconazole-N-Oxidem/z 336.1 -> 143.1

Voriconazolem/z 350.1 -> 127.0

Posaconazolem/z 701.3 -> 127.0

Hydroxyitraconazolem/z 721.3 -> 408.2

Itraconazolem/z 705.3 -> 392.2

CompoundRange

(µg/mL)LLOQ

(µg/mL)%RSD

at LLOQTotal


Fluconazole 0.5–100 0.3750 13.1 ≤2.7% ≤2.6%Hydroxyitraconazole 0.05–10 0.0500 18.8 ≤11.5% ≤10.0%

Itraconazole 0.05–10 0.0375 17.2 ≤8.9% ≤8.6%Posaconazole 0.05–10 0.0500 15.2 ≤7.7% ≤5.2%Voriconazole 0.05–10 0.0250 15.8 ≤2.6% ≤1.5%

Voriconazole N-Oxide 0.05–10 0.0375 16.1 ≤5.4% ≤3.5% Table 1. Performance characteristics of the analytes evaluated. Range defined by linear fit where r2 >0.99. LLOQ defined by S/N (PtP) >10 and %RSD ≤20%. % RSD at LLOQ determined through analytical sensitivity experiments performed over three occasions (n=30). Total precision and repeatability of QCs performed over five occasions in stripped serum (n=25).



CONCLUSIONSThe Waters ACQUITY UPLC I-Class/Xevo TQD IVD System has demonstrated the capability to deliver an analytically sensitive and precise method for fluconazole, hydroxyitraconazole, itraconazole, posaconazole, voriconazole, and voriconazole-N-oxide in serum.



EXPERIMENTAL DETAILSThe ACQUITY UPLC I-Class/Xevo TQD IVD System was controlled by MassLynx™ IVD (v4.1) and the data processed using the TargetLynx™ Application Manager. Calibrators and Quality Controls were prepared by spiking commercially available reference material in plasma and the samples were processed using the following conditions:

Sample preparation conditions50 µL sample was processed in a liquid-liquid extraction with acidified ethyl acetate followed by a solvent exchange to 0.1% formic acid in water prior to analysis.

LC conditionsColumn: ACQUITY UPLC HSS PFP

1.8 µm, 2.1 mm × 100 mm

Mobile phase A: Water

Mobile phase B: Acetonitrile


Gradient: 2% B for 0.5 minutes, 2–60% B over 1.5 minutes, 98% B for 0.5 minutes, 98% A for 0.5 minutes



Polarity: ESI (-)


ACQUITY UPLC I-Class/Xevo TQD IVD System: Analytical Performance for a Thymidylate Synthase Inhibitor


This document describes a test of the analytical performance of the ACQUITY UPLC I-Class/Xevo TQD IVD System for the analysis of 5-fluorouracil in plasma.

RESULTSPerformance characteristics of busulfan on the ACQUITY UPLC I-Class/Xevo TQD IVD System are shown in Table 1. A chromatogram illustrating the analytical sensitivity of the busulfan analysis is shown in Figure 1.

Compound Range (ng/mL)

LLOQ (ng/mL)

%RSD at

LLOQ


5-fluorouracil 20–2000 7.5 20.0 ≤9.0% ≤7.2% Table 1. Performance characteristics of 5-fluorouracil. Range defined by linear fit where r 2 >0.99. LLOQ defined by S/N (PtP) >10 and %RSD ≤20%. %RSD at LLOQ determined through analytical sensitivity experiments performed over five occasions (n=50). Total precision and repeatability of QCs performed over five occasions in plasma (n=25).

24 Therapeutic Drug Monitorng





Figure 1. Chromatogram showing the analysis of 5-fluorouracil using the ACQUITY UPLC I-Class/Xevo TQD IVD System.

20 ng/mL 5-fluorouracil calibrator m/z 129.0>41.9

Plasma blank


CONCLUSIONSThe Waters ACQUITY UPLC I-Class/Xevo TQD IVD System has demonstrated the capability to deliver analytical sensitivity and precision for the analysis of 5-fluouroracil in plasma.


Return to Index

25Therapeutic Drug MonitorngReturn to Index


EXPERIMENTAL DETAILSThe ACQUITY UPLC I-Class/Xevo TQD IVD System was controlled by MassLynx™ IVD (v4.1) and the data processed using the TargetLynx™ Application Manager. Whole blood Calibrators and Quality Controls were processed using the following conditions:

Sample preparation conditions50 µL sample was processed with aqueous zinc sulphate, acetonitrile, and centrifuged. Each analyte was analyzed individually.

LC CONDITIONSColumn: ACQUITY UPLC HSS C18 SB

1.8 µm, 2.1 mm x 30 mm




Gradient: 50% B for 0.2 minutes, 50–100% B over 0.4 minutes, hold 100% B for 0.6 minutes, equilibrate with 50% B for 0.6 minutes at 0.6 mL/min


ACQUITY UPLC I-Class/Xevo TQD IVD System: Analytical Performance for Immunosuppressive Agents


This document describes a test of the analytical performance of the ACQUITY UPLC I-Class/Xevo TQD IVD System for the analysis of cyclosporine, everolimus, sirolimus, and tacrolimus in whole blood.



Polarity: ESI (+)

RESULTSPerformance characteristics of cyclosporine, everolimus, sirolimus, and tacrolimus on the ACQUITY UPLC I-Class/Xevo TQD IVD System are shown in Table 1. Analytical sensitivity of the system is illustrated in Figure 1.

Return to Index



Waters, The Science of What’s Possible, ACQUITY, UPLC, Xevo, MassLynx, and TargetLynx are trademarks of Waters Corporation. All other trademarks are the property of their respective owners.

©2018 Waters Corporation. Produced in the U.S.A. November 2018 720006425EN AG-PDF

Figure 1. Chromatogram showing the analysis of 25 ng/mL cyclosporine and 1 ng/mL everolimus, sirolimus, and tacrolimus using the ACQUITY UPLC I-Class/Xevo TQD IVD System.

CompoundRange

(ng/mL)LLOQ

(ng/mL)%RSD

at LLOQTotal


EQA mean bias

Cyclosporine 25–1500 5 7.0 ≤5.7% ≤1.8% -0.2%Everolimus 1–30 0.5 6.5 ≤7.7% ≤4.7% -11.9%Sirolimus 1–30 1 12.7 ≤9.1% ≤6.1% -6.1%

Tacrolimus 1–30 0.5 17.5 ≤6.3% ≤2.6% +1.6% Table 1. Performance characteristics of cyclosporine, everolimus, sirolimus, and tacrolimus. Range defined by linear fit where r2 >0.99. LLOQ defined by S/N (PtP) >10 and %RSD ≤20%. % RSD at LLOQ determined through analytical sensitivity experiments performed over five occasions (n=50). Total precision and repeatability of QCs performed over five occasions in whole blood (n=25). EQA mean bias determined by comparison of obtained values to the LC-MS all laboratories trimmed mean (LC-MS ALTM) value (n=33). Note: The EQA mean bias for everolimus (-11.9%) is based on the returned EQA results for just two laboratories.



CONCLUSIONThe Waters ACQUITY UPLC I-Class/Xevo TQD IVD System has demonstrated the capability to deliver analytical sensitivity, accuracy, and precision for the analysis of cyclosporine, everolimus, sirolimus, and tacrolimus in whole blood.

Tacrolimus (m/z 821.5>768.5) 8.53e3

4.69e3

2.64e3

4.37e3

1 ng/mL

Sirolimus (m/z 931.6>864.5) 1 ng/mL

Everolimus (m/z 975.6>908.6) 1 ng/mL

Cyclosporine (m/z 1219.8>1202.8) 25 ng/mL

Tacrolimus (m/z 821.5>768.5) 8.53e3

4.69e3

2.64e3

4.37e3

1 ng/mL

Sirolimus (m/z 931.6>864.5) 1 ng/mL

Everolimus (m/z 975.6>908.6) 1 ng/mL

Cyclosporine (m/z 1219.8>1202.8) 25 ng/mL


Waters, The Science of What’s Possible, ACQUITY, UPLC, MassLynx, TargetLynx, Xevo, VanGuard, CORTECS, and Oasis are trademarks of Waters Corporation. All other trademarks are the property of their respective owners.

©2019 Waters Corporation. Produced in the U.S.A. April 2019 720006366EN IH-PDF

Documents

Clinical LC-MS/MS Systems: Analytical Capabilities · 2019. 4. 15. · 17-OHP 0.76–303 0.76 9.2% ≤8.2% ≤8.2% -5.0% DHEAS 140–54000 140 7.0% ≤3.9% ≤2.7% 5.8% Table 1. Performance