msacl practical training section 010818...1/8/2018 2 illicit drug use by women 2011‐2012 • 5.9...

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Method Development Case History: The continuous need for improvements in laboratory developed tests (LDTs) with a

broad drug panel for neonatal drug testing as a case example

Stephanie J. Marin, Ph.D.

Biotage, LLC

Part 1: Proof of concept, method development and validation

Outline for Part 1:

• Neonatal drug testing

• Umbilical cord tissue

• Proof of Concept

• Nicotine and metabolites in meconium and cord tissue

• Method Development

• LC‐TOF‐MS method

• Homogenization

• Sample preparation and clean up

• Method validation

• Comparison with outside laboratory

• Study with meconium results and chart review

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Why neonatal drug testing?

• Identify neonates exposed to drugs in utero for management of

• intoxication• dependence and withdrawal• long‐term needs (social and medical)

• Child custody issues

3

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Illicit Drug Use by Women

2011‐2012

• 5.9 percent of pregnant women were illicitdrug users (10.7 non‐pregnant)

• 18.3% aged 15 to 17

• 9% aged 18 to 25

• 3.4% aged 26 to 44

• Increased from 2009‐2010 results (4.4%, 16.2%, 7.4%, 1.9%)

Substance Abuse and Mental Health Services Administration, Results from the 2012 National Survey on Drug Use and Health: Summary of National Findings, NSDUH Series H‐46, HHS Publication No. (SMA) 13‐4795. Rockville, MD: Substance Abuse and Mental Health Services Administration, 2013. 4

Specimens for in utero drug exposure

Umbilical Cord Tissue• Forms by the fifth week

• Two arteries, one vein encased in Wharton’s jelly

• Drug distribution not well known

• Every child has one

• Typically about 20 in long

• Easy to collect at birth

Meconium• Forms 12‐16 weeks gestation

• The “gold standard” to detect in utero drug exposure

• Drug distribution well studied

• Can be expelled in utero

• Limited amount of specimen

• Can take several days to collect

5

Umbilical Cord Challenges...

• Cord must be prepared/homogenized• Drugs must be extracted• Sample clean‐up• Low level drugs must be detectedNot as easy as it sounds

Can we do this?Cord Tissue Challenges

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Proof of ConceptNicotine and Metabolites in Meconium – Cord Tissue

• 19 paired meconium and umbilical cord specimens

• Smoking habits and known second‐hand exposure for each trimester• Smoked consistently (3‐20 cigarettes/day) during the second and third trimesters, n = 14

• Stopped smoking during pregnancy, n = 2 (1 each 1st and 2nd trimesters)

• Exposed to second‐hand smoke only during pregnancy, n = 3

• Analysis by LC‐MS/MS method• 0.25 g meconium, 2 g cord tissue

• Extracted and analyzed in duplicate

• Nicotine, cotinine, 3‐OH‐cotinine

Marin SJ, Christensen RD, Baer VL, Clark CJ, McMillin GA, Ther Drug Monit, 33:80‐5, 2011 7

y = 0.0107x + 1.6973R² = 0.2803

0

2

4

6

8

10

0 100 200 300 400 500 600

cord tissue (ng/g)

meconium (ng/g)

Nicotine

y = 0.5165x + 17.267R² = 0.8367

0

50

100

150

200

250

300

350

0 50 100 150 200 250 300 350

cord tissue (ng/g)

meconium (ng/g)

Cotinine

y = 0.4701x + 19.876R² = 0.8425

0

50

100

150

200

250

300

350

400

0 100 200 300 400

cord tissue (ng/g)

meconium (ng/g)

3‐OH‐Cotinine

Marin SJ, Christensen RD, Baer VL, Clark CJ, McMillin GA, Ther Drug Monit, 33:80‐5, 2011

Proof of ConceptPaired Meconium – Cord Results

ng/g mec cord mec cord mec cord

mean 129.1 4.6 71.1 48.09 104.5 61.29

median 79.9 3.6 44.7 29.24 79.0 56.92

max 590.1 15.0 317.5 157.71 354.2 195.25

min <4.0 <0.5 <2.0 <0.25 <4.0 <0.5

nicotine 3‐OH‐cotininecotinine

8

• 67 analyte qualitative panel validated and online for serum and plasma

• Accurate mass data from high resolution time‐of‐flight MS

• Two “calibrators” at cutoff to separate isobars

• Two positive controls at 150% cutoff, negative control

• Three deuterated markers

• Automatic column regeneration to increase throughput

• Full scan allows retroactive data review

• Drug ‐metabolite pairs increases confidence in results

9

DevelopmentQualitative LC‐TOF‐MS panel

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Positivity criteria based on:• Retention time• Mass error• Isotope abundance and spacing• TOF “score”• Area counts


Example Data

Morphine, Oxymorphone, Hydromorphone…other opioids

Stimulants (Amps, Cocaine)Benzos

THC

Naloxone, Naltrexone

Barbiturates

THC

Overlaid EICs positive mode Overlaid EICs negative mode


• Tissue‐miser• “Dremel” type device

• Fritsch Ball Mill• Geno grinder

• Jar with SS beads• Vigorously shaken

• Covaris Cryo‐prep• Freeze in liquid nitrogen• “Hammer”

Homogenized in methanol, dried in CVE*, reconstituted

*CVE = centrifugal vacuum evaporation

DevelopmentCord Homogenization

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• Tissue‐miser• Fritsch Ball Mill• Geno grinder• Covaris Cryo‐prep

DevelopmentCord Homogenization

Strata X, Strata X Drug‐B (Phenomenex), XCEL (UCT)

DevelopmentCord Extraction SPE

Product Pre‐treatment Wash Elute

Strata X water water 15:85 EtOAc:IPA

Strata X water water, 10% MeOH 15:85 EtOAc:IPA



Strata X water water ACN

Strata X Drug‐B pH 5 acetate water, 10% MeOH 1)15:85 EtOAc:IPA, 2)7:2:1 EtOAc:IPA:NH4OH



Strata X Drug‐B pH 5 acetate pH 5 acetate 1)15:85 EtOAc:IPA, 2)7:2:1 EtOAc:IPA:NH4OH

XCEL 0.1 M phosphate pH 6 0.1 M acetic acid, hexane 50:50 EtOAc:hexane, 98:2 EtOAc:NH4OH

MeOH = methanolEtOAc = ethyl acetateIPA = isopropanolNH4OH = ammonium hydroxide

14

Supported Liquid Extraction (SLE+, Biotage)• Load, wait, elute• Pre‐treatment: water, 0.5M NH4OH• Elute: ethyl acetate, dichloromethane

Toxi‐tubes (Agilent)• Pre‐treatment: methanol + water, water• Extraction: sample is rocked, L/L extraction, organic layerremoved and dried

QuEChERS (Agilent)• Pre‐treatment: reconstituted 9:1 water:methanol• Followed manufacturer’s directions

DevelopmentCord Extraction

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Strata X and X Drug‐B• No significant difference with different MeOH washes

• Good recovery for most drugs

Xcel• Poor recovery

Biotage SLE+• Water better than 0.5 M NH4OH

• EtOAc better than dichloromethane

• Extracts were extremely clean

DevelopmentCord Extraction Results

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Toxi‐tubes

• Reasonable recovery

• Too many manual steps

• No longer available

Quechers

• No difference if MeOH present

• Good recovery but manual process

DevelopmentCord Extraction Results

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Cord Extraction ResultsHypnotics

Strata X Drug‐B and Xcel samples were injected in duplicate

nAverage

Scoren

Average

Scoren

Average

Scoren

Average

Scoren

Average

Scoren

Average

Score

Benzodiazepine 24 74 48 78 28 83 22 61 23 79 48 82

Alprazolam 3.0 2 86 4 72 2 90 4 87

Chlordiazepoxide 3.0 2 48 4 35 2 54 2 85 4 56

Clonazepam 3.0 2 62 4 77 4 88 2 58 2 68 4 98

Diazepam 3.0 2 69 4 85 4 82 2 56 2 82 4 99

Flunitrazepam 3.0 2 63 4 75 4 76 2 59 2 49 4 64

Flurazepam 3.0 2 74 4 98 2 57 2 97 4 81

Lorazepam 3.0 2 89 4 88 4 83 2 83 2 82 4 96

Midazolam 3.0 2 98 4 81 2 60 2 90 4 94

Nitrazepam 3.0 2 74 4 81 4 68 2 53 2 41 4 85

Oxazepam 3.0 2 86 4 88 4 90 2 89 2 84 4 96

Temazepam 3.0 2 70 4 70 4 94 2 39 1 94 4 53

Triazolam 3.0 2 70 4 90 2 65 2 87 4 71

Benzodiazepine metabolite 16 82 30 82 8 90 14 74 14 87 32 87

2‐Hydroxyethylflurazepam 3.0 2 97 4 92 4 91 2 53 2 89 4 95

7‐Aminoclonazepam 3.0 2 90 4 96 2 92 2 97 4 92

7‐Aminoflunitrazepam 3.0 2 62 4 75 2 86 2 93 4 80

alpha‐Hydroxyalprazolam 3.0 2 77 4 91 2 90 4 78

alpha‐Hydroxymidazolam 3.0 2 64 4 55 2 57 2 71 4 60

alpha‐Hydroxytriazolam 3.0 2 96 4 95 2 78 2 77 4 98

Desalkylflurazepam 3.0 2 95 4 86 4 89 2 88 2 93 4 93

Nordiazepam 3.0 2 73 2 65 2 66 4 99

Z Drugs 3.0 3 45 7 82 4 92 4 79 7 93

Zolpidem 3.0 2 59 4 91 2 89 2 94 4 97

Zopiclone 3.0 1 31 3 74 2 94 2 64 3 90

Totals ‐ 43 74 85 80 36 84 40 69 41 82 87 85

Biotage SLE+

Drug Class/CompoundCutoff

(ng/g)

Strata X Strata X Drug B Xcel Toxi‐tubes QuEChERS

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nAverage

Scoren

Average

Scoren

Averag

e Scoren

Average

Scoren

Average

Scoren

Averag

e Score

Opioid ‐ 21 52 51 71 11 34 20 63 19 78 42 70

Buprenorphine 0.6 1 31 4 67

Codeine 1.3 2 55 4 60 4 61 2 81 2 66 4 84

Fentanyl 0.1 4 86 4 10 2 87 4 65

Hydrocodone 1.3 2 50 4 74 2 78 2 82 1 73

Hydromorphone 1.3 2 36 4 93 2 84 2 90 4 73

Meperidine 0.6 2 50 4 54 2 45 2 64 4 58

Methadone 3.0 2 66 4 51 3 32 2 36 1 51 4 60

Morphine 1.3 2 57 4 84 2 74 2 73 4 67

Oxycodone 1.3 3 41 2 40 2 74

Oxymorphone 1.3 2 51 4 64 2 79 3 68

Propoxyphene 3.0 2 60 4 80 2 87 4 58

Tapentadol 0.6 2 58 4 86 2 57 2 89 4 82

Tramadol 0.6 2 60 4 84 2 54 2 90 4 75

Opioid Antagonist ‐ 4 45 8 87 4 95 4 82 8 78

Naloxone 2.5 2 34 4 81 2 93 2 81 4 91

Naltrexone 2.5 2 56 4 94 2 98 2 83 4 65

Opioid metabolite ‐ 18 54 36 72 3 27 14 69 20 77 40 73

6‐monoacetyl‐morphine 1.3 2 56 4 96 2 94 2 93 4 91

Buprenorphine‐3‐glucuronide 0.6 4 90 2 73 4 70

Dihydrocodeine 1.3 2 82 4 45 2 34 2 99 2 61 4 92

EDDP 3.0 2 77 4 96 2 91 4 86

N‐desmethyltramadol 0.6 2 51 4 52 1 19 2 57 2 80 4 60

Norbuprenorphine 1.3 1 18 4 92 2 65 2 54

Norbuprenorphine‐3‐glucuronide 2.5 1 35 3 48 2 67 4 57

Norfentanyl 0.1 2 56 4 70 2 71 2 64 2 60

Normeperidine 0.6 2 63 3 70 2 39 2 88 4 92

Norpropoxyphene 3.0 2 42 2 66 2 29 4 62

O‐desmethyltramadol 0.6 2 62 2 90 2 87 4 84

Totals ‐ 43 52 95 73 14 31 38 68 43 78 90 72

QuEChERS Biotage SLE+


(ng/g)

Strata X Strata X Drug B Xcel Toxi‐tubes

Cord Extraction ResultsAnalgesics

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nAverage

Scoren

Average

Scoren

Average

Scoren

Average

Scoren

Average

Scoren

Average

Score

Stimulant 18 65 43 77 8 45 21 58 24 88 41 62

Amphetamine 2.5 4 81 2 49 2 85 4 60

MDA 2.5 2 60 3 76 4 36 2 60 2 88 4 60

MDEA 2.5 2 58 2 71 2 97 4 76

MDMA 2.5 2 64 4 77 2 84 2 94 4 55

Methamphetamine 2.5 2 60 4 85 2 56 2 83 4 66

Methylphenidate 2.5 2 72 4 84 2 56 2 94 2 61

Phentermine 2.5 4 88 2 47 2 89 1 34

Ritalinic Acid 2.5 2 64 4 60 1 39 2 83 2 27

Cocaine 2.5 2 62 4 59 2 56 2 98 4 89

Benzoylecgonine 2.5 2 84 4 90 2 52 2 97 4 83

Cocaethylene 2.5 2 63 4 77 4 53 2 70 2 91 4 70

m‐Hydroxybenzoylecgonine 2.5 4 69 2 62 4 59

Hallucinogen 3.0 2 67 6 67 3 42 4 84 4 69

11‐nor‐delta‐9‐Carboxy‐THC 7.5 2 53 3 42 2 78

Phencyclidine (PCP) 1.3 2 67 4 81 2 89 4 69

Totals ‐ 20 65 49 75 11 44 21 58 28 88 45 62

QuEChERS Biotage SLE+


(ng/g)

Strata X Strata X Drug B Xcel Toxi‐tubes

Cord Extraction ResultsStimulants and Hallucinogens

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DevelopmentSLE+ Optimization

• Methanol and CVE vs water or water with 0.1% Triton X‐100

• Cord/liquid ratio

• Ethyl acetate vs 90:10 ethyl acetate:isopropanol

• 2 vs 3 elution fractions

Can eliminate methanol, extract cord in water, Triton X‐100 improved recovery for some drugs

• TOF issues, switched to water only

1 g cord/2 mL water, load 1 mL (SLE+ column capacity)

2 x 2.5 mL fractions of 90:10 EtOAc:IPA21

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Umbilical Cord Challenges...

• Cord must be prepared/homogenized

• Drugs must be extracted

• Sample clean‐up

• Low level drugs must be detected

…and solutions

• Homogenized by freezing in liquid nitrogen and pulverizing

• Extracted with 0.1% Triton X‐100 in water

• Centrifuged and supernatant undergoes supported liquid extraction

• Analysis by LC‐TOF‐MS

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Can we do this?Cord Tissue Challenges

Cord Cutoffs (ng/g)

• barbiturates: 20‐40

• benzodiazepines and metabolites: 5‐10

• cocaine and metabolites: 8

• PCP: 4

• zolpidem: 10

• methadone/EDDP: 10

• opioids and metabolites: 1‐10

• opioid antagonists: 8

• stimulants: 8

ValidationCord Tissue Cutoffs (ng/g)

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ValidationPatient Specimen Correlation

• 32 patient specimens analyzed by an outside laboratory• Immunoassay screen with reflex to GC‐MS or

LC‐MS/MS

• TOF detected all 42 positive results found by the outside laboratory

• TOF found 12 additional compounds and 64 additional positive results

• Analytes not included in outside lab panel = 18

• Metabolites of previously detected compounds = 19

• TOF only positive results with drug and metabolite = 21

Marin SJ, Metcalf A, Krasowski MD, Linert BS, Clark CJ, Strathmann FG, McMillin GA, Ther Drug Monit,36:119‐24, 2014 24

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1 concentrations reported by outside laboratory

2 compounds confirmed by outside laboratory and detected by TOF

3 drugs and metabolites detected by TOF not reported by the outside laboratory

4 compounds not included in outside laboratory assay

Drug or MetaboliteConc

(ng/g)1Detected by Both Labs2

Additional Compounds

Detected by TOF3Drug or Metabolite Conc (ng/g)1 Detected by

Both Labs2

Additional Compounds

Detected by TOF3

neg - 2 meperidine >20 1methamphetamine >50 2 1 tramadol 2

amphetamine 5.3 to >50 2 1 n-desmethyltramadol4 1benzoylecgonine (Be) 2.4 to >10 3 norpropoxyphene 2

cocaine4 1 alprazolam 5.4 to 11.9 3 1

m-OH-Be4 1 alpha-OH-alprazolam4 3

codeine 3 clonazepam4 2

hydrocodone 3.5 to >20 5 5 7-aminoclonazepam4 3

dihydrocodeine4 7 diazepam 3.5 1 1morphine 8.5 to 10.6 2 2 nordiazepam >20 1 4

hydromorphone 2 to 7.3 3 4 oxazpam 1oxycodone 8.1 to >20 6 2 temazepam 1

oxymorphone 3.1 to 3.6 2 3 midazolam 4 1 2

methadone >20 5 alpha-OH-midazolam4

EDDP 7 to >20 3 2 zolpidem 7fentanyl 2 Total 42 64

Marin SJ, Metcalf A, Krasowski MD, Linert BS, Clark CJ, Strathmann FG, McMillin GA, Ther Drug Monit,36:119‐24, 2014

ValidationPatient Specimen Correlation

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ValidationPaired Meconium‐Cord Data

• 57 cord specimens, 37 paired with meconium results

• Chart review info

• 44 negative specimens• 26 paired, 18 no meconium

• 2 had prescriptions for drugs not in ARUP assay

• 5 positive meconium specimens• 3 positive for opioids – 1 negative in cord

• 2 positive for 9‐carboxy‐THC (not in ARUP assay)

• 5 negative meconium, but found compounds by TOF

Marin SJ, Metcalf A, Krasowski MD, Linert BS, Clark CJ, Strathmann FG, McMillin GA, Ther Drug Monit,36:119‐24, 2014 26

Meconium Results Cord Results Chart Review

Morphine 2 ng/g, codeine 72 ng/g morphine, codeine APAP/codeine 1 day before birth

Morphine 87 ng/g, oxycodone 5 ng/g morphine, oxycodone

Negative diazepam, oxycodoneHas had prescriptions for both both but no documented use in pregnancy

Negative fentanyl Fentanyl for procedure 1 day before birth

Negative zolpidem Zolpidem 2 days prior to delivery

Negative zolpidemZolpidem 1 day before delivery, previous oxycodone prescription

Negative zolpidem Zolpidem 1 day prior

Hydrocodone 17 ng/g, hydromorphone 19 ng/g, dihydrocodeine-qual only, codeine 135 ng/g, oxycodone 2 ng/g, oxymorphone 8 ng/g

neg History of drug abuse

Marin SJ, Metcalf A, Krasowski MD, Linert BS, Clark CJ, Strathmann FG, McMillin GA, Ther Drug Monit,36:119‐24, 2014

ValidationPaired Meconium‐Cord Data

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Validated to meet CAP, CLIA and NYDOH standards

Ease of collection, ample specimen

No waiting for meconium to pass

Identifies 58 specific drugs and metabolites by LC‐TOF‐MS

Faster TAT

Covaris Cryoprep provided the best cord homogenate and high throughput with minimal risk of contamination

SLE+ provided an easy, fast, elegant solution for clean up of extracted cord tissue

SummaryCord Tissue Method

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SummaryCord Tissue Method

Cryoprep bags very expensive, require liquid nitrogen

Distribution of drugs and metabolites in cord tissue not fully known or understood

Concentrations of drugs analytes in cord tissue lower than in meconium

May detect drugs administered during labor and delivery

Qualitative panel with reporting based on area counts

Two LC injections/sample

“Go live” August 2012…. and wait….

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Gwen McMillin, Ph.D., DABCC(CC,TC)

University of Utah and ARUP Laboratories

Part 2: Can’t you do this faster and better? Understanding and addressing needs of technical staff and clinicians

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Outline for Part 2:

• Technical needs:

• Managing growth

• Consistency from batch to batch, instrument to instrument

• Dealing with poorly performing analytes

• Clinical needs:

• Faster time to result

• Improved positivity rates for some drugs

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The test started to grow rapidly…

Where are the opportunities?

• Sample preparation• One at a time handling too slow• Safety concerns• Hard to secure enough blank cord

• Analysis• Instrument capacity becoming a problem

• Is it necessary to retain two injections per sample?

• Data Interpretation• Variable area counts, variation in the “score”• Variation in QC performance• Cutoffs don’t seem consistent – what is “real”?

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Old sample preparation

• Slice cord

• Place ~1 g in TT2 bag, attached to a 5mL tube

• Place TT2 bag in liquid nitrogen

• Place TT2 bag in Covaris Cryprep and “hammer” 3x’s

• Turn bag around and repeat “hammer”

• Remove bag and tap tissue into tube before it thaws

• Add deuterated markers and 0.1% Triton X‐100

• Add 1/8 tsp 0.9‐1.5mm stainless steel beads and secure caps

• Place tubes in Bullet Blender and homogenize twice for 5 min

• Incubate for 1 hour

• Centrifuge at 14K rpm and 0°C for 15 min

• Transfer 1 mL supernatant onto 6mL SLE+ columns

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New sample preparation

• Slice cord

• Place ~1 g in TT2 bag, attached to a new 5mL tube

• Place TT2 bag in liquid nitrogen

• Place TT2 bag in Covaris Cryprep and “hammer” 3x’s

• Turn bag around and repeat “hammer”

• Remove bag and tap tissue into tube before it thaws

• Add deuterated markers and 0.1% Triton X‐100 5 µg/mL DNase

• Add 6 large UFO (5.6 mm)1/8 tsp 0.9‐1.5mm stainless steel beads and secure caps

• Place tubes in Bullet Blender and homogenize twice for 5 min

• Incubate for 1 hour

• Centrifuge at 14K rpm and 0°C for 15 min

• Transfer 1 mL supernatant onto 6mL SLE+ columns

35

Performance was comparable

36

Patient results were qualitatively equivalent for four days of parallel runs, although area counts varied

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Bulk production of drug‐free matrix

• Never enough blank! • Frequent lot changes contributed to variation in performance of the assay

• Solution:• Selected drug free cords and determined wet weight• Lyophilized• Divided lyophilized weight by wet weight to determine how much would be equivalent to 1 g cord (~0.07 g)

• Pre‐weighed in sample preparations • Reconstituted as needed for use as calibrators and controls

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Monitoring clinical performance: positivity rates

• University of Iowa study suggested similar positivity rates in cord vs meconium (Palmer et al., Clin Biochem 50:255‐61, 2017)

• Clients frequently called to complain about false negatives for buprenorphine

• Decided to report if area counts exceed 5000 and score >64

• Look at adding norbuprenorphine

• Vanderbilt study suggested that detection of some opioids was a problem...

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BUPRENORPHINE

Cord

1.8%

Meconium

5.2%

AMPHETAMINE 9.0% 8.2%

METHADONE 4.3% 4.4%

OPIATES 9.5% 13.6%

BENZODIAZEPINES 4.7% 3.2%

BENZOYLECGONINE 3.3% 4.4%

BARBITURATES 2.9% 1.1%

Example of clinical concerns

Vanderbilt University (n=217 newborns)• Agreement varied based on drug class; 104 discordant pairs

• 17 cord positive, meconium negative

• 45 cord negative, meconium positive

• 42 cord positive, meconium positive but for different analytes

• Many NAS infants had negative cord tests

• Conclude that cord collection was logistically preferred but that cord was not as sensitive as meconium for several clinically important opioids

Colby, Clin Biochem 50:784‐90, 2017

How can we achieve better sensitivity (lower cutoffs) for key analytes?39

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14

41

LC‐MS/MS

• Fragmentation to potentially offer improved specificity and sensitivity

• Conversion to “semi‐quantitative” results by normalization with deuterated markers (“internal standards”) and use of a single‐point calibrator forced through the origin

• QC at 50% and 150% of cutoff (plus a negative)• Switch to 1 injection and streamlined chromatography reduced anaytical time from 11.5 to 5.0 min

• Data interpretation and QC became more consistent

Haglock‐Adler CJ, McMillin GA, Strathmann FG, Clin Biochem 49:1092‐5, 2016

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Added more “deuterated markers”• Morphine‐d3, 1.03 min

• Methamphetamine‐d5, 1.69

• 6‐AM‐d6, 1.71

• Benzoylecgonine‐d3, 2.10

• Meperidine‐d4, 2.29 min

• Phenobarbital‐d5, 2.47

• A‐OH‐alprzaolam‐d5, 3.18

• Diazepam‐d5, 3.51

Changed up the compounds

• Added• Chlordiazepoxide

• Methylphenidate

• N‐desmethyl‐Tapentadol

• Norbuprenorphine

• Noroxycodone

• Noroxymorphone

• Norhydrocodone

• Normeperidine

• Ritalinic acid

• Omitted• Flunitrazepam

• 7‐Aminoflunitrazepam

• Flurazepam

• Nitrazepam

• Triazolam

• Naltrexone

• MDEA

• MDA

44

Patient comparison overview

514 patient samples were compared between the TOF and LC‐MS/MS methods, covering 38 drug analytes

• 260 were negative by TOF, 259 negative by LC‐MS/MS (1 hydrocodone)

• 12 were negative by LC‐MS/MS and positive by TOF: 8 were clonazepam

• 9 were positive by LC‐MS/MS and negative by TOF due to mass shift errors and poor scores

• 504 compounds were detected by both TOF and LC‐MS/MS but ~60% fell below the quantitative cutoffs

So we had to validate new cutoffs

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Examples of new cutoffsParent drug Target analytes and

change to cutoffsOld cutoff (ng/g) New cutoff (ng/g)

May, 2016

Oxycodone Oxycodone 4 0.5

Noroxycodone 4 1

Oxymorphone 4 0.5

Noroxymorphone 4 0.5

Buprenorphine Buprenorphine 2 1

Norbuprenorphine 8 0.5

Buprenorphine glucuruonide

8 1

Cocaine Cocaine 8 0.5

Benzoylecgoninie 8 0.5

M‐hydroxy‐benzoylecognine

8 1

Cocaethylene 8 146

Established better criteria for reporting

47

Example data from new LC‐MS/MS method

48

Live Feb, 2016!

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Monitoring positivity rates per client

49

Monitor QC, qualitatively and “semi”‐quantitatively…

• In validation 1,231 results were evaluated for each QC • 50% CV’s ranged from 16.8% (alprazolam) to 49.2% (methadone)

• 150% CV’s ranged from 15.9% (triazolam) to 51.7% (zopiclone)

• 2.5% of results were qualitatively incorrectly

• In production, we monitor/trend monthly• Quantitative: CVs

• Qualitative: % rejections

A switch in deuterated markers…

From benzoylecognine‐d3 to meperidine‐d4 for associated analytes

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After 16 revisions to the SOP,we still have work to do…

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Simuli Wabuyele, Ph.D.

ARUP Institute for Clinical and Experimental Pathology

Part 3: Re‐development and validation

Outline for Part 3:

• Method Improvement

• Matrix‐matched blank cord

• Production of bulk blank cord

• Homogenization

• LC‐MS/MS

• Sample preparation

• Method Transfer: Agilent Platforms

• 6460 to 6470

• Method Validation

• Qualitative assay

• Method comparison

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Re‐develop due to change

Re‐validate due to change

Method in Routine use

Change the Method

Evaluate Effect

Modified from Chauhan et al., J Anal Bioanal Tech 2015

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Method ImprovementMatrix‐Matched Blank Cord

55

• Lyophilized drug‐free cord • Prepared in‐house at ARUP only

• Cost and complex equipment

• Concerned it may be too different

• Fresh drug‐free cord• More representative of the patient sample matrix

Method ImprovementProduction of Bulk Blank Cord• De‐identify residual umbilical cord specimens

• Screen by LC‐MS/MS method

• Pool about 80 ‐100 drug‐free cords

• Chop each by hand!

• How about a mini food chopper ?• Freeze pooled drug‐free cords

• Place them in the food chopper

• Push a button!

Inexpensive Less time consuming User friendly

Bait cutter

Aliquoted and stored frozen (‐80°C) until use

Method ImprovementHomogenizationBullet Blender

• Worked well until volume increase• Top rotor malfunction

• Breaking tubes

• Inconsistent homogenization

Bead Ruptor 24

Freeze samples (‐80°C) for 15 min.prior to homogenization

Homogenization Challenges• Heat production• Stability of drug analytes

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Sample PreparationSupported Liquid Extraction (SLE)

58

Aqueous sample is absorbed onto sorbent ‘no flow through’ Collect and dry eluate

Matrix components

SLE Support material (diatoms)

Analyte

STEP 1: Load 1 mL cord supernatant

STEP 2: Wait for 5 min STEP 3:

Elute with organic solvent (2.5 mL x 2)

Apply positive pressure

Wait 5 minApply pressure

http://www.biotage.com/news/isolute‐sle‐is‐an‐excellent‐option

Problems with the Assay

• Particulates in extracted samples

• Interferences and poor chromatography

• Frequent re‐extractions• Time‐consuming

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Problems Caused

60

Early elutersaffected

~35 patients re‐extracts

QC

Fail

Sample loop clogging

Changed 1‐2 days

Spin vials

4k rpm max

LC needle bottom sensing turned off

Band‐Aid solutions

Inspect samples

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Balancing Resources

Method Improvement

SLE: production method

Method re‐development was needed for better performance of the assay

LC‐MS/MS Method Improvements

• Deuterated markers (most had intensities of 10^7)• Decrease concentration by dilution

• Introduced a guard column cartridge

• HPLC column • Changed from 5.0 to 2.6 µm ID• Evaluated different vendors

• Phenomenex Phenyl‐hexyl (50 x 3mm i.d.)

• Optimize MRM mass transitions• Quantifier and qualifier

Goals• Extend LC‐MS lifetime• Increase sensitivity• Increase specificity

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Compound NameProduction Method Transitions (m/z) Optimized Mass Transitions (m/z)

Precursor Ion Product Ion Precursor Ion Product Ion

A‐Hydroxymidazolam 342.1324 Water Loss

342.1168

203 203

Lorazepam 321302.9 Water Loss

321229

275 275

Methamphetamine 150.1119.2

Same Transition 150.1119.2

119.1 91

Naloxone 328.2310.1 Water Loss

328.2268

212 253

Noroxycodone 302.1284.1 Water Loss

302.1227

187 187

Noroxymorphone 288.1270.1 Water Loss

288.1173

213 213

O‐Desmethyl‐Tramadol 250.258.3

Same Transition 250.230.2

58.2 58.2

Oxycodone 316.2298.1 Water Loss

316.2256.1

241 241.1

Oxymorphone 302.1284.1 Water Loss

302.1198

227 227

Temazepam 301.1283 Water Loss

301.1176.9

255 255

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Sample Preparation

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SELECTIVITY

• Solid Phase Extraction (SPE)

• Supported Liquid Extraction (SLE)

• Dilute and Shoot

Complex procedureHighly SelectiveCleaner extracts

Simple procedureLess SelectiveDirtier extracts

• Diatomaceous earth Compare with synthetic SLE

• Evaluate different elution solvents

• Mixed‐mode cation exchange Strata XLC Oasis MCX Evolute Express CX, Isolute HCX

Improvements

0%

20%

40%

60%

80%

100%

120%

140%

160%

Biotage 90/10 EtoAC:IPA (Production) Biotage 95/5 EtOAC:IPA Novum 95/5 EtOAc:IPA

Diatomaceous earth vs Synthetic SLE

Evaluation of SLE Elution Solvents

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Mixed‐mode Cation Exchange SPE Procedure

67

Pre‐treat sample: 1:1.5 0.1M Phosphate buffer pH 6.0

Condition: 2 mL MeOH

Equilibrate: 2 mL 0.1M Phosphate buffer pH 6.0

Load pretreated sample

Wash 1 : 1 mL 0.1M Phosphate buffer pH 6.0

Wash 2 : 1 mL 0.1M Acetic acid (optimal)

Dry at 30 psi for 6 min

Add 1 mL hexane to remove residual water and dry

Elute 1: 60:40 Ethyl acetate:hexane (600 µL x 2)

Wash 3 : 1 mL 98:2 Methanol:acetic acidDry for 1 min

Elute 2 : 78:20:2 DCI:IPA:NH4OH (600 µL x 2)LC‐MS/MS Analysis

Add 1% HCL in methanol andEvaporate under N2 @ 37°C

Reconstitute in 100 µL (90:10 Water:methanol)

0%

20%

40%

60%

80%

100%

120%

Evolute 60mg CX 3cc Isolute HCX 200mg 3cc

Oasis MCX 3cc Strata XLC 60mg 3cc

Various SPE Columns Evaluated

0%

10%

20%

30%

40%

50%

60%

70%

80%

50:50 Ethyl acetate:hexane 60:40 Ethyl acetate:hexane

SPE Extraction Efficiencies (%,n=4)

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Method Validation Qualitative Assay• Instrument: Agilent 6460 Triple Quad LC‐MS

• Calibration• Single calibrator (Cutoff)

• Linear regression force through zero

• Controls : Negative, 50% and 150% the cutoff

• Prepared in pooled drug‐free umbilical cord tissue

• 7 Deuterated analogs were used as internal standards

50%

150%

Calibrator (Cutoff)

Imprecision (%CV) and extraction recovery (%RE) were determined

Method comparison was performed o Comparison of SPE vs SLE (current method in production)

o 98 patient specimen and 10 proficiency testing (PT) samples were used

Method Validation Average Recovery and Imprecision (n=6)

Compound RE 50% RE 150% CV 50% CV 150% Compound RE 50% RE 150% CV 50% CV 150%

6‐acetylmorphine 52.0% 57.3% 18.9% 12.6% Methadone 43.3% 64.3% 23.8% 8.1%

7‐aminoclonazepam 51.3% 48.6% 16.9% 13.4% Methamphetamine 60.8% 65.2% 4.8% 3.0%

Alprazolam 38.9% 41.7% 19.9% 21.2% m‐OH‐benzoylecgonine 33.0% 32.0% 9.5% 9.7%

Amphetamine 58.2% 60.9% 3.8% 7.8% Midazolam 39.1% 45.8% 12.1% 13.3%

Benzoylecgonine 48.6% 47.2% 5.9% 5.5% Morphine 47.6% 40.5% 15.0% 7.6%

Buprenorphine Glucuronide 4.5% 3.5% 53.6% 32.5% N‐Desmethyl Tramadol 53.8% 54.1% 10.5% 22.7%

Buprenorphine 16.2% 19.2% 36.2% 21.9% Norbuprenorphine 56.2% 64.8% 40.6% 37.9%

Butalbital 58.0% 57.0% 4.8% 3.5% Nordiazepam 35.0% 41.8% 11.6% 8.3%

Clonazepam 46.1% 38.6% 14.4% 5.7% Norhydrocodone 56.0% 60.6% 13.5% 25.3%

Cocaine 52.2% 56.9% 7.8% 5.2% Noroxycodone 52.4% 50.1% 39.0% 12.2%

Codeine 52.2% 48.8% 7.7% 22.4% Noroxymorphone 41.2% 43.8% 7.8% 36.5%

Diazepam 41.3% 46.5% 3.9% 2.3% O‐Desmethyl‐Tramadol 55.8% 58.1% 8.0% 18.0%

Dihydrocodeine 56.4% 55.2% 14.3% 22.7% Oxycodone 65.6% 64.2% 12.7% 21.8%

ⱡEDDP 35.2% 41.6% 20.3% 22.9% Oxymorphone 50.7% 49.8% 9.2% 29.8%

Fentanyl 47.1% 86.6% 21.3% 18.2% Tramadol 54.2% 59.0% 3.9% 5.4%

Hydrocodone 58.3% 59.5% 16.0% 26.2% Zolpidem 53.6% 57.1% 9.3% 5.9%

Hydromorphone 58.3% 53.7% 15.0% 26.0% ⱡ2‐ethylidene‐1,5‐dimethyl‐3,3‐diphenylpyrrolidine (EDDP)

Method Comparison98 Authentic Patient Specimen• Negative samples : 35 SLE / 35 SPE

• 33 Individual compounds identified

Drug/Metabolite Class # of Patient Samples SPE + SLE+Missed SPE ‐

MissedSLE ‐

Amphetamine Stimulants 11 9 11 2

None

Benzoylecgonine Stimulants 13 13 13Cocaine Stimulants 5 4 5 1Methamphetamine Stimulants 10 9 10 1m‐Hydroxybenzoylecgonine Stimulants 3 2 3 1

Total 42 37 42 5


MissedSLE ‐

7‐aminoclonazepam Sedatives/hypnotics 1 1 1

NONE!

Alprazolam Sedatives/hypnotics 4 4 4Butalbital Sedatives/hypnotics 4 4 4Clonazepam Sedatives/hypnotics 1 1 1Diazepam Sedatives/hypnotics 2 2 2Midazolam Sedatives/hypnotics 2 2 2Nordiazepam Sedatives/hypnotics 2 2 2Zolpidem Sedatives/hypnotics 2 2 2

Total 18 18 186‐acetylmorphine abused/other 2 2 2

Total 2 2 2

+SLE/‐SPE: Low drug levels overall, possible stability issues?

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Method Comparison Cont’dDrug/Metabolite Class # of Patient Samples SPE + SLE+

Missed SPE ‐

MissedSLE ‐

Buprenorphine Glucuronide Analgesics 9 6 9 3 0Buprenorphine Analgesics 4 4 4Codeine Analgesics 2 2 1 0 1Dihydrocodeine Analgesics 1 1 1EDDP Analgesics 5 4 5 1 0Fentanyl Analgesics 7 6 6 1 1Hydrocodone Analgesics 3 2 2 1 1Hydromorphone Analgesics 3 3 3Methadone Analgesics 4 4 4Morphine Analgesics 7 7 7N‐Desmethyl Tramadol Analgesics 1 1 1Norbuprenorphine Analgesics 20 19 20 1 0Norhydrocodone Analgesics 4 3 3 1 1Noroxycodone Analgesics 4 4 4Noroxymorphone Analgesics 3 3 2 0 1O‐Desmethyl‐Tramadol Analgesics 1 0 1 1 0Oxycodone Analgesics 2 2 2Oxymorphone Analgesics 1 1 1Tramadol Analgesics 1 1 1

Total 82 73 77 9 5

‐SLE/+SPE: SLE ion suppression, SPE better sensitivity

Method Comparison10 Proficiency Testing (PT) samples


MissedSLE ‐

Buprenorphine Glucuronide Analgesics 4 3 4 1 0

Buprenorphine Analgesics 3 2 3 1 0

Norbuprenorphine Analgesics 5 3 5 2 0EDDP Analgesics 1 1 1

Methadone Analgesics 1 1 1

Morphine Analgesics 1 1 1

Noroxycodone Analgesics 3 3 2 0 1

Noroxymorphone Analgesics 4 4 4

Total 22 18 21 4 1Alprazolam Sedatives/Hypnotics 1 1 1

Total 1 1 1

Amphetamine Stimulants 1 1 1

Benzoylecgonine Stimulants 1 1 1

Methamphetamine Stimulants 1 1 1

m‐OH‐benzoylecgonine Stimulants 1 1 1

Total 4 4 4

PT samples analyzed by SPE a month later (NOT IDEAL) – Low drug levels detected by SPE possible stability issues

Method TransferAgilent Platforms: 6460 to 6470

Slope : 0.957Intercept : ‐1.165R= 0.9940Deming SEE: 10.87

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26

LDTs Life Cycle continues….

Development

ValidationOptimization

What did we learn?

• Do the best you can; recognize that what works for low volume assays may not work as volume changes

• Continually monitor assay performance, looking for technical improvement opportunities

• Communicate with production lab staff and stay apprised of their practical issues and needs

• Listen to clients to understand clinical needs

• Re‐develop and re‐validate based technical, practical and clinical requirements, being open to new approaches

77

Acknowledgments

78

• ARUP Toxicology Lab, with key contributors:

• Chantry Clark• Anna Metcalf• Brandy Hill• Noah Flint• Carrie Haglock‐Adler• Triniti L. Scroggin• Fred Strathmann, Ph.D., DABCC

• University of Iowa• Intermountain Healthcare

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Thank you!

80

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