1525-1635 a fudge.ppt

Vitamin DVitamin D

800 samples a day by LC-MS/MS

For our patients and our population

• The measurement of high volume analytes utilizing the specificity y g p yconferred by liquid chromatography coupled with mass spectroscopy is an coupled with mass spectroscopy is an idea whose time has come!


Why LC MS/MS?Why LC-MS/MS?

• Quality of the results–LC-MS/MS method at FMCLC MS/MS method at FMC–Specificity

Accuracy–Accuracy• IMVS had long standing interest in

calcium metabolism, osteoporosis and vitamin Dand vitamin D

• Costs


How?How?• Tender processp• Complete system

800 l d• 800 samples a day• Working applicationWorking application

–SimpleRobust–Robust

–Commercial calibrators etc• Capital costs• Low ongoing costs


• Low ongoing costs

TLX-4 plus Quantum Access MAX


MultiplexingMultiplexingP k • Peaks are sent to the detector for only 25% of only 25% of the total run time leaving it idle 75% it idle 75% or the time.


MultiplexingMultiplexing


TLX 4TLX-4


Sample PreparationSample Preparation

• Simple protein precipitation• Including addition of deuterated • Including addition of deuterated

internal standardDi tl i t 96 ll l t f • Directly into 96 well plates for ease of automation

• Autosampler samples from centrifuged 96 well platescentrifuged 96 well plates


Hamilton StarletHamilton Starlet


96 well plates96 well plates


Sample CleanupSample Cleanup

• On-line• Turboflow columns• Turboflow columns• Interfering substances trapped• Sample focused onto analytical

columncolumn• Turboflow column cleaned up with

o ganic sol entsorganic solvents


ChromatographyChromatography

• Accucore C18 50 x 3 mm 2.6u• Methanol gradient• Methanol gradient• Method time including sample

i j ti l di t l ti t injection, loading step, eluting step and re-equilibration 6.5 mins.

• X4 = sample every 1.6 mins800 sample in 22 ho s• 800 sample in 22 hours


Mass SpectroscopyMass Spectroscopy

• TSQ Quantum Access Max• APCI ionization in positive mode• APCI ionization in positive mode• Monitor three MRN transitions per

l tanalyteParent Q3 Q3 Q3Parent Q3 Q3 Q3

25OH D3 383.4 105.2 257.2 365.425OH D3 383.4 105.2 257.2 365.4

25OH D2 395.4 209.1 210.9 377.3

25OH D3-d6 389.3 211.0 263.3 371.3

25OH D2 395.4 209.1 210.9 377.3

25OH D3-d6 389.3 211.0 263.3 371.3


ChromatogramsChromatograms35681201 - TIC - RT: 0.03 - 1.00 NL: 7.40E4F: + c APCI SRM ms2 383.393 [105.150-105.170, 257.160-257.180, 365.430-365.450]

95

100 RT: 0.53

35681201 - TIC - RT: 0.02 - 1.00 NL: 1.56E5F: + c APCI SRM ms2 389.300 [210.990-211.010, 263.290-263.310, 371.260-371.280]

95

100 RT: 0.52

55

60

65

70

75

80

85

90

95

nten

sity

55

60

65

70

75

80

85

90

nten

sitySample D3 88 nmol/L Internal Standard

10

15

20

25

30

35

40

45

50

Rel

ativ

e In

0.930.96

0.35 0.66 10

15

20

25

30

35

40

45

50

Rel

ativ

e In

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9Time (min)

0

5

100.730.47 0.830.07 0.15 0.280.21 0.78

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9Time (min)

0

5

10

0.10 0.930.63 0.810.720.580.300.22 0.880.42 0.980.380.16

36486377 - TIC - RT: 0.05 - 1.00 NL: 5.82E4F: + c APCI SRM ms2 395.365 [209.090-209.110, 210.960-210.980, 377.243-377.263]

100 RT: 0.55

55

60

65

70

75

80

85

90

95

nsity

S l D2 55 l/L

15

20

25

30

35

40

45

50

55

Rel

ativ

e In

ten

0.60

Sample D2 55 nmol/L


0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0Time (min)

0

5

100.760.29 0.880.700.340.14 0.38 0.490.10 0.79 0.96

Assay performanceAssay performanceUTAK QC Pools

Low Level 1 Level 2 D3 L 1 1 2 3

D3 D2 D3 D2 D3 D2 D3 D3 D3 D3

mean 28.0 24.5 78.0 74.2 180 178 25.3 13.1 70.2 142

SD 3.30 3.10 7.50 8.27 13.5 19.1 2.94 1.00 5.72 8.39

CV% 11.8 12.7 9.6 11.2 7.5 10.7 11.6 7.6 8.1 5.9

n 36 36 36 36 36 36 36 30 30 30n 36 36 36 36 36 36 36 30 30 30

Stated value

25 24.2 74.9 72.7 182 177 25

%recovery 112 101 104 102 99 101 101


Routine PrecisonRoutine PrecisonLC MS/MS ISYS

Low Level 1 Level 2 poolMean 27 7 76 4 177 6 73 5

pool69 2

LC-MS/MS ISYS

Mean 27.7 76.4 177.6 73.5SD 2.49 4.82 9.33 4.12

CV% 9.0 6.3 5.3 5.6

69.24.696.8

n 200 200 200 165Target 27.0 74.9 182.2

200

% rec 103 102 97


LoQLoQ

• Estimated using CLSI guidelines• 30 samples with low values• 30 samples with low values• Run in duplicate over 6 days• 20% CV used.

Accucore Accucore > 1500 inj

Kinetex

25-OH D3 5.2 8.0 6.625 OH D3 5.2 8.0 6.625-OH D2 5.0 9.8 5.0


Accuracy DEQASAccuracy - DEQAS

100

110

70

80

90

MS Identity

50

60

IMVS

- LC

MSM

Passing & Bablok (I) f it(2.04 + 0.93x)

95% CI bands

20

30

40

1010 20 30 40 50 60 70 80 90 100 110

DEQAS LCMS


Routine DEQASQNIST reference Measurement

Sample No 3-epi D2 D3 D2+D3 D2+D3+epi ALTM IMVS%bias from

D2+D3421 2.33 0.96 57.50 58.5 60.83 55.4 60.6 3.6%422 1.70 1.66 36.58 38.2 39.90 36.2 37.2 -2.6%423 5.68 0.98 84.62 85.6 91.28 81.2 84.6 -1.2%424 2.58 0.98 46.20 47.2 49.78 47.0 45.0 -4.7%425 2 50 0 96 46 18 47 1 49 60 46 9 46 5 1 3%425 2.50 0.96 46.18 47.1 49.60 46.9 46.5 -1.3%426 0.40 1.06 33.40 34.5 34.90 32.0 32.4 -6.1%427 4.50 0.88 75.20 76.1 80.60 72.5 74.2 -2.5%428 2.80 2.45 52.30 54.8 57.60 51.8 50.7 -7.5%429 3.00 0.50 58.90 59.5 62.50 58.5 52.2 -12.3%430 0.80 22.3 17.60 39.9 40.70 35.0 42.7 7.0%431 1.20 1.25 22.60 23.9 25.10 24.9 26.0 8.8%432 2.90 2.69 48.60 51.3 54.20 50.0 51.5 0.4%433 11.70 1.18 90.40 91.6 103.30 88.2 94.0 2.6%434 2.40 4.42 74.20 78.6 81.00 67.2 79.2 0.8%435 2.40 0.53 46.10 46.6 49.00 45.8 53.8 15.5%436 4 00 1 25 76 70 78 0 82 00 83 2 83 6 7 2%436 4.00 1.25 76.70 78.0 82.00 83.2 83.6 7.2%437 1.40 1.38 33.30 34.7 36.10 35.2 36.8 6.1%438 2.70 1.81 54.80 56.6 59.30 57.3 62.2 9.9%439 2.30 1.18 39.70 40.9 43.20 42.6 44.2 8.1%


440 2.30 1.31 47.10 48.4 50.70 51.3 53.9 11.4%2.2%

Linearity D3Linearity D325-OH Vitamin D3

Linearity Plot

450

300

350

400

Linear f it

200

250

300

Obt

aine

d

(1.047 +0.9953x)

50

100

150

00 50 100 150 200 250 300 350 400 450

Expected


Linearity D2Linearity D225-OH Vitamin D2

Linearity Plot

400

300

350

Linear f it

150

200

250

Obt

aine

d

(0.6114 +1x)

50

100

150

00 50 100 150 200 250 300 350 400

Expected


IssuesIssues

• Reliability–Experience and more preventative Experience and more preventative

maintenance is helping –Monitoring overnight runsMonitoring overnight runs

• Turn around times


TrainingTraining

• Start with good people• Get the supplier to train as many as • Get the supplier to train as many as

practical first handTh h d i t i i• Thorough and ongoing training

• Key staff sharing experienceKey staff sharing experience• Document the learning experience• Don’t be afraid to use a spanner and

a screwdriver


Key Learnings!Key Learnings!

• Capacity• Applications support• Applications support• Service engineering support• All suppliers must “walk the walk”

not just “talk the talk” when it comes not just talk the talk when it comes to the clinical marketYOU CAN DO IT!• YOU CAN DO IT!


AcknowledgmentsAcknowledgments• Dr Peter O’LoughlinDr Peter O Loughlin• Dr Malcolm Whiting• Dr Peter Galettis• Shane McDonnell –Thermo• Shane McDonnell Thermo• Phil Wilson – Thermo• Cass Stuart• Carrie Tran• Carrie Tran• Pascale Dubois


• Narelle Burke

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