The new MassChrom® diagnostics kit (orderNo. 57000) for determining amino acids and acyl-carnitines differs from the previous product (orderNo. 55000) due to extremely simple specimen pre-paration, characterised by the omission of derivati-sation. The analytes are extracted directly from thefilter card. High precision is ensured by measuringin MRM mode. Stable isotope-marked internal stan-dards are also used. This kit variant has been de-veloped with particular focus on rapid phenyl-ketonuria diagnosis (The Editor).

The measurement of amino acids and acylcarni-tines from dried whole blood spots on filter paperusing LC-MS/MS has become a standard method inthe early detection of treatable congenital diseasesas a part of newborn screening (see issue 1/07 ). Themajor advantage of thismethod is the possibi-lity of semi-quanti-tatively recording arange of diagnosticallyrelevant metabolites ofseveral substance classesfrom only around 3 µlwhole blood in onesingle measurement.The convent iona lsample preparation in-volves the derivatisati-on of analytes and in-ternal standards to butylesters. This takes appro-ximately 2.5 to 3 hours.The measurement resultitself is available appro-ximately 2 minutes afterinjecting the preparedsample. This work re-ports on the resultsobtained with the newdirect amino acid andacylcarnitine measure-ments without priorderivatisation.

Material and methods

All measurementsare carried out using aWATERS LC-MS/MS sys-tem, consisting of the

Alliance 2795 HPLC with autosampler and the "Quat-tro Micro" tandem MS. While injecting samplesdirectly (20 µl) without prior chromatographic sepa-ration, a flow gradient with a reduced flow of30 µl/min is applied during the scanning interval.All analytes and internal standards are measured inMRM mode (MRM = Multiple Reaction Monitoring).This method guarantees maximum selectivity withhigh sensitivity, but necessitates the careful tuningof each analyte. The underlying mass transitions andinternal standards are listed in Table 1."Neolynx" software calculates the analyte concen-trations based on the deuterated internal standards(kit art. No. 57004). Results are then evaluated aseither "inconspicuous" or "conspicuous". The referencevalues used are our own statistically determinedreference range limits ("cut off", 99.9th or 0.1st per-

w w w . c h r o m s y s t e m s . d e

1/08LOGDIANew shorter Sample Preparation

Amino Acids andAcylcarnitinesNon DerivatisedDr. rer. nat. Marina Stopsack, University Hospital Carl Gustav Carus Dresden, Hospital andOutpatient Clinic for Paediatric and Adolescent Medicine, Metabolism and Screening Laboratory

Page 1/2/3

Amino Acids and Acylcarnitines Non DerivatisedDr. Marina Stopsack, University Hospital Carl GustavCarus Dresden

Page 3

MassChrom® Amino Acids and Acylcarnitinesfor Newborn ScreeningProduct information

Page 4

Newborn Screening at LiègeBoemer François, CHU Sart-Tilman,University of Liège, Belgium

Page 5

MassTox® Psychotropic DrugsGregor Schütze, Dr. Stephan Burghardt, Chromsystems

Page 6

MassTox® Antidepressants and Neuroleptics withTandem-MSProduct information

Page 6

“www.chromsystems.de“ now with EQASparticipations and overviews on calibrators

Page 7/8

Combined Analysis Vitamins B1/B6-Analysiswith Internal StandardDr. Wiebke Großberger, Chromsystems

Page 8

NEW: Combined Analysis of Vitamin B1 (wholeblood) and Vitamin B6 (whole blood/plasma/serum)Product information

Page 8

Chromsystems increases production capacitiesand invests in research and development

Page 9

Clinical relevance of pyridinium crosslinksDr. Richard Lukacin, Chromsystems

Crosslinks in UrineProduct information

Page 10

TDM in a regional hospital:C.H.R. de la Citadelle in LiègeThierry Gougnard, Jean-Marc Minon

Page 11

Solutions for Demanding Clinical Analysis atApplied Biosystems/MDS Analytical TechnologiesLisa Sapp, Product Manager Applied Biosystems

Page 12

New workshops for diagnosticsby LC-MS/MS

Dates, Imprint

Analyte Abbr. Mass transition Int. Standard

Glycine GLY 76 > 30 13C215N-GLY

Alanine ALA 90 > 44 D4-ALAValine VAL 118 > 72 D8-VALLeucine/isoleucine LEU 132 > 86 D3-LEUMethionine MET 150 > 104 D3-METPhenylalanine PHE 166 > 120 D5-PHECitrulline CIT 176 > 70 D2-CITTyrosine TYR 182 > 136 D4-TYRFree carnitine C0 162 > 85 D9-C0Acetylcarnitine C2 204 > 85 D3-C2Propionylcarnitine C3 218 > 85 D3-C3Butyrylcarnitine C4 232 > 85 D3-C4(Iso)valerylcarnitine C5 246 > 85 D9-C5Tiglylcarnitine C5:1 244 > 85 D9-C5Glutarylcarnitine C5DC 276 > 85 D6-C5DCHexanoylcarnitine C6 260 > 85 D3-C6Octanoylcarnitine C8 288 > 85 D3-C8Decanoylcarnitine C10 316 > 85 D3-C10Decenoylcarnitine C10:1 214 > 85 D3-C10Dodecanoylcarnitine C12 344 > 85 D3-C12Myristoylcarnitine C14 372 > 85 D3-C14Myristoleylcarnitine C14:1 370 > 85 D3-C14Tetradecadienoylcarnitine C14:2 368 > 85 D3-C14Palmitoylcarnitine C16 400 > 85 D3-C16Hydroxypalmitoylcarnitine C16OH 416 > 85 D3-C16Stearoylcarnitine C18 428 > 85 D3-C18Octadecenoylcarnitine C18:1 426 > 85 D3-C18Hydroxystearoylcarnitine C18OH 444 > 85 D3-C18Hydroxyoctadecenoylcarnitine C18:1OH 442 > 85 D3-C18

Table 1: Mass transitions of the analytes and internal standards

ChromSystemS ®

centile of the analyte's frequency distribution forneonates with an age of > 36 hours to 5 days and amaturity of > 32 weeks' gestation).The sample preparation can be significantly simplifiedwith the principle applied for all analytes: The lyophi-lised internal standard is reconstituted according tothe manufacturer's specifications. For each sample,a blood spot with a diameter of 3.2 mm is punchedinto a microtitre plate and 200 µl standard workingsolution is pipetted into each well. The microtitreplate is covered with aluminium foil and agitatedfor 20 minutes at room temperature and 650–750rpm. On completion of incubation, the plate is cen-trifuged to sediment eluted specimen substrate fibresand is subsequently analysed and evaluated.

Validation of the non-derivatising method

The suitability of the method is assessed accor-ding to general analytical principles based on impre-cision, inaccuracy, linearity and detection limit andalso with regard to the requirements of newbornscreening (samples are not guaranteed to be homo-geneous, therefore semi-quantitative; but conspicuousspecimens are considerably higher than normal speci-mens). To achieve this, external dry blood qualitycontrols were used in different concentration ranges.Depending on concentration, the intra-assay precisionfor amino acids lies between 1.9 and 8.5 %, and foracylcarnitines between 4.9 and 7.6 % at the relevantreference range limit.In the same concentration range, the inter-assayprecision of amino acids was determined to be bet-ween 7.3 % and 10.6 %, and that of acylcarnitines7.2 and 17.2 %. The inaccuracy for the metabolitetracers of the newborn screening target diseases variesbetween -12.4 % and 4.1 % (amino acids) as well-12.3 % and 16.0 % (acylcarnitines). These deviationsare taken into consideration by the use of device-specific, statistically determined reference ranges (asdescribed above) obtained from the measurement ofinternal specimens. This method is confirmed bysuccessful participation in two (to date) internationalEQAS tests for amino acids and acylcarnitines in dryblood.All measured analytes (two times five specimens each)were correctly classified. Refer to the overview fordetails (Tab. 2).The linearity of the measurements was assessed basedon the recovery of the declared value of positive dryblood specimens. The correlation coefficient for themetabolite tracers of the newborn screening targetdiseases is 0.991 to 1.000 (see Fig. 1).

Clinical application examples

The two most frequent newborn screening meta-bolic defects, which are recorded with tandem MS,are PKU (phenylketonuria: Phenylalanine hydroxy-lase) and MCAD (medium-chain acyl-CoA dehydro-genase defect). In 2005, 172 newborns were affectedby these in Germany, 29 were suffering from anothertarget disease recorded with tandem MS and 289other diagnoses were verified using 4 other mono-

analyte screening methods . In this case, it is signifi-cant that the level of the metabolite concentrationis extensively dependent on the actual metabolicsituation.On the third day of life, the optimal time for screening,degradation of molecules occurs. While high concen-

trations of medium-chain acylcarnitines must beexpected in the case of MCAD, problems might ariseif the blood sample is taken or checked significantlylater. The metabolic situation may since have changedand levels of octanoylcarnitine (C8) may have nor-malised in extreme cases, despite MCAD (Fig. 2).Both the derivatised and the non-derivatised methodguarantee sufficiently fast analysis times for this; theproblem in this case primarily involves the reportingroute and specimen transportation.In addition to newborn screening, the analysis ofamino acids and acylcarnitines is becoming increa-singly important in selective diagnostics in clinicallysymptomatic newborns and older children. Extremelysimple sample preparation, the speed of measurementand the parallel recording of many analytes makeLC-MS/MS from dry blood, in the described form,an extremely meaningful method. Two clinicalexamples serve to make this clear:

1. A two-day old newborn with hyperammonaemia (NH4+

= 300 µmol/l). The risk of irreversible brain damageincreases along with the level and duration of the ammo-nium concentration. A very high citrulline concentration

Page 21/08LOGDIA

Fig. 1: Recovery of amino acids and acylcarnitines in enriched dry blood specimens

Specimen Certification, Year: 2007, Quarter: 4, expected values*

Amino AcidsAnalyte Specimen Specimen Specimen Specimen Specimen

4751 4752 4753 4754 4755Result Code Result Code Result Code Result Code Result Code

Phe (mg/dl blood) 7,7 2 1,4 1 1,0 1 1,3 1 0,8 1Leu (mg/dl blood) 3,0 1 3,3 1 2,5 1 2,5 1 8,2 2Met (mg/dl blood) 0,5 1 4,0 2 0,4 1 0,5 1 0,2 1Tyr (mg/dl blood) 1,1 1 1,5 1 9,3 NE 1,4 1 8,9 NEVal (mg/dl blood) 1,9 1 2,1 1 1,5 1 2,1 1 7,1 2Cit (mg/dl blood) 0,6 1 0,4 1 0,5 1 3,4 2 0,2 1

NE = Clinical assessment not evaluated

AcylcarnitinesAnalyte Specimen Specimen Specimen Specimen Specimen

4761 4762 4763 4764 4765Result Code Result Code Result Code Result Code Result Code

C3 (µmol/l blood) 1,52 1 2,32 1 1,20 1 2,05 1 18,68 2C4 (µmol/l blood) 0,21 1 0,25 1 6,13 2 5,26 2 0,52 1C5 (µmol/l blood) 0,14 1 0,15 1 5,10 2 0,13 1 0,27 1C5DC (µmol/l blood)** 0,89 2 0,08 1 0,07 1 0,04 1 0,04 1C6 (µmol/l blood) 0,04 1 2,05 2 0,03 1 0,03 1 0,03 1C8 (µmol/l blood) 0,05 1 10,09 2 0,05 1 0,04 1 0,05 1C10 (µmol/l blood) 0,06 1 1,61 2 0,06 1 0,05 1 0,06 1C14 (µmol/l blood) 0,11 1 0,12 1 4,13 2 0,10 1 0,15 1C16 (µmol/l blood) 0,92 1 1,08 1 20,75 2 0,83 1 1,22 1

*Sum of endogenous and enrichment values, **CDC assayed values

Data Verification and Evaluation, Year: 2007, Quarter: 4 Lab: 549

Amino AcidsAnalyte Specimen Specimen Specimen Specimen Specimen

4751 4752 4753 4754 4755Result Code Result Code Result Code Result Code Result Code

Phe (mg/dl blood) 7,8 2 1,6 1 1,1 1 1,5 1 1,2 1Leu (mg/dl blood) 2,5 1 3,1 1 2,1 1 3,4 1 8,5 2Met (mg/dl blood) 0,3 1 3,6 2 0,2 1 0,4 1 0,2 1Tyr (mg/dl blood) 1,4 1 2,1 1 9,2 2 1,9 1 10,3 2Val (mg/dl blood) 1,8 1 2,2 1 1,5 1 2,2 1 8,0 2Cit (mg/dl blood) 0,6 1 0,6 1 0,7 1 3,7 2 0,5 1

AcylcarnitinesAnalyte Specimen Specimen Specimen Specimen Specimen

4761 4762 4763 4764 4765Result Code Result Code Result Code Result Code Result Code

C3 (µmol/l blood) 1,63 1 1,83 1 1,33 1 1,85 1 22,64 2C4 (µmol/l blood) 0,21 1 0,21 1 5,97 2 5,31 2 0,54 1C5 (µmol/l blood) 0,13 1 0,14 1 5,31 2 0,14 1 0,33 1C5DC (µmol/l blood) 5,67 2 0,07 1 0,08 1 0,09 1 0,08 1C6 (µmol/l blood) 0,02 1 1,90 2 0,02 1 0,02 1 0,02 1C8 (µmol/l blood) 0,06 1 10,97 2 0,06 1 0,05 1 0,05 1C10 (µmol/l blood) 0,05 1 1,66 2 0,06 1 0,05 1 0,06 1C14 (µmol/l blood) 0,06 1 0,05 1 3,20 2 0,06 1 0,10 1C16 (µmol/l blood) 0,95 1 1,03 1 21,99 2 0,89 1 1,34 1

Codes: 1 = Within normal limits 2 = Outside normal limits*= No quantitative data reportedREVIEWER'S COMMENTS, EVALUATION: No misclassifications were reported – 100% Satisfactory.

Tabelle 2: Ergebnisse des Ringversuches Aminosäuren und Acylcarnitine [2]

is measured in the amino acid mass spectrum (see Fig. 3).The cause of the hyperammonaemia is a citrulline de-gradation disorder in the urea cycle.

2. Eight-year old child, admitted to hospital in a comawith hypoglycaemia of 0.3 mmol/l. The presence of afatty acid oxidation defect is primarily suspected. Theamino acid mass spectrum reveals generalised, extensivehyperaminoacidaemia (see Fig. 4a). In contrast, theacylcarnitines are inconspicuous to low. The cause wasexogenous hepatic coma as a result of mushroom poisoning.

In both cases, the results for the amino acids andacylcarnitines in dry blood without derivatisationwere available within one hour of the specimens'arrival at the laboratory. As a result of this, bothchildren were diagnosed very rapidly and providedwith specific therapy.

Summary

The determination of amino acids and acylcarni-tines without prior derivatisation in MRM mode withChromsystems kit components can be achievedwithout technical problems. The package size, shelflife and handling of the kit components support this.The new, easier sample preparation contributes tothe speed of LC-MS/MS measurement. The omissionof the derivatisation reaction saves time and costs,

and simplifies the screening. All key analytical varia-bles convincingly meet the requirements of semi-quantitative screening analyses using dry blood.Experience with the direct measurement of aminoacids and acylcarnitines in MRM mode has beenacquired over several years, and over a six-monthperiod using the above described kit components.

Literature:

[1] Ceglarek, U, Neugeborenenscreening mit Tandem-Massenspektrometrie, DIALOG Chromsystems 1/07, 1–3

[2] Newborn Screening Quality Assurance Program, CDC Atlanta:http://wwwn.cdc.gov/nsqap/Public/default.aspx

[3] Deutsche Gesellschaft für Neugeborenenscreening, Jahresreport 2005, S. 12:http://www.screening-dgns.de/PDF/Screeningreport_2005.pdf

Page 31/08LOGDIA

Fig 2b: MCAD 4.2 days old

C8

Fig 2c: Normal spectrum of acylcarnitines

C8

Fig 3a: Patient specimen, citrullinaemia

CIT

Fig 3b: Normal spectrum of citrulline/ornithine

CIT

Fig 4a: Non-specific, generalised hyperacidaemia

ALA

LEU

PHE

Fig 4b: Normal spectrum of amino acids

PHE

LEU

ALA

C8

Fig. 2a: MCAD 2.1 days old

PRODUCT INFORMATION

Sample preparation withoutderivatisation

Acylcarnitines

Amino Acids

Order number 57000

> All required reagents and isotope labelledstandards included

> Dried blood spot controls included> Direct sample injection without derivati-

sation> High selectivity and sensitivity by ms/ms

detection

Sample preparation:• Punch out 3 mm blood spots from the filter

card into a well plate• Add 100 µl Internal Standard,

agitate 20 min• Inject 10 µl of extract into the LC-MS/MS

system

MassChrom®

Amino AcidsandAcylcarnitinesfor NewbornScreening

New: Analysis of Succinyl Acetone

(Diagnosis of Tyrosinaemia)

The Biochemical Genetics Laboratory, locatedat the University Hospital of Liège (Belgium), is oneof the six accredited Belgian Centres for NeonatalScreening. Our laboratory has more than fortyyears experience in diagnosing phenylketonuria.Meanwhile, the outlook has been broadened withscreening programmes for galactosemia, congeni-tal hypothyroidism, cystic fibrosis, congenital ad-renal hyperplasia, biotinidase and a1-antitrypsinedeficiency, sickle cell disease and glucose-6-phosphate dehydrogenase deficiency. About17,000 newborns are tested each year for thesedifferent pathologies.

Expanded screening implementation

For more than ten years, amino acid and acyl-carnitine analysis of newborn dried blood spots usingtandem mass spectrometry (TMS) has been rapidlygaining worldwide support for screening for metabolicdiseases. Numerous reports in literature illustrate itsutility for detection of amino, organic, and fatty aciddisorders [1–4]. More than 30 disorders can be dia-gnosed at birth [5–6] and, thus, preventive medicalcare can be initiated for affected neonates. Quanti-tative acylcarnitine profiles can also be examined inchildren or adults with clinical symptoms or familyhistories suggestive of these disorders.Considering that, in 2006, our laboratory acquireda tandem mass spectrometer to implement screeningfor amino acid and acylcarnitine disorders, we initiallychose to validate a homemade derivatised method,applying neutral loss and parent scan acquisitionmodes. Next, in order to improve sensitivity and toreduce our relative high false positivity rate for somecarnitine derivates, we modified our mass strategyto a full MRM acquisition mode and finally, at theend of 2007, we decided to switch to a commercialsolution, facilitating correspondence between ourlaboratory and actual quality requirements.Many reasons led us to opt for a ChromSystemsarticle: A competitive price and the TÜV SÜD certi-fication were primary arguments in our choice. Later,the simplification of our batch traceability, the goodvalidation results we harvested and the efficientafter-sales service all contributed to our satisfaction.This newborn screening kit has been the first productwe purchased from Chromsystems and since trusthas rapidly grown, based on our 8-month experiencewithout troubles, other Chromsystems products havealready been introduced to our laboratory (i. e. kitfor CDT in serum).

Positive experiences with results

Up to now, we have diagnosed two MSUDpatients and one PKU patient out of 5,500 samplestested with this kit. Concentrations of leucine/isoleucine observed for the two MSUD patients were1,250 and 1,262 µmol/l (normal population P50 =179 µmol/l, cut-off = 451 µmol/l). Valine was mea-sured at 361 and 239 µmol/l respectively (normalpopulation P50 = 91 µmol/l, cut-off = 315 µmol/l).The amino acid MRM profiles of one of the firstMSUD patients and one normal patient are proposedin Figure 1.This first MSUD patient left the maternity unit onday 5 without clinical manifestations. On day 10,she was hospitalised at the intensive care neonatologyunit with severe dehydration, hypoglycaemia, keton-uria and hypertonic episodes. Neonatal screeningresults were available on day 12 and indicated an

important increase of leucine/isoleucine levels.Critical haemodialysis was immediately initiatedallowing a significant decrease in leucine concentra-tion (from 3308 µmol/l to 391 µmol/l in 24 hours).Diagnosis confirmation was made based on aminoacid chromatography in combination with organicacid analysis. Thiamine was also administered overseveral weeks. Now, the four month-old girl is growingup normally with leucine levels being checked threeto four times per week and a favourable outcomemay reasonably be expected. In this case, althougha maple syrup urine disease was initially suspectedon a clinical basis, the neonatal screening resultswere the first to clearly identify the disease.

Quality improvement

We have not yet diagnosed any disorder asso-ciated with an abnormal acylcarnitine profile. Dueto the low incidence of the screened diseases the

laboratory needs to participate at external qualitycontrol (QCE) programmes.Quarterly, the CDC (Center for Disease Control andPrevention) sends, free of charge, several kinds ofmaterial for proficiency testing and quality controlof the amino acid and acylcarnitine tests(www.cdc.gov).

In Europe, the ERNDIM (European Research Networkfor Evaluation and Improvement of Screening,Diagnosis and Treatment of Inherited MetabolicDisorders) can also provide material for proficiencytesting of acylcarnitine profiles. In practice, normalor pathological specimens are distributed two orthree times per year to the participating laboratories.Final reports are sent to participants informing themof the reliability of their results (www.erndimqa.nl).

Additionally, the Region4Genetics collaborativeproject led by Dr Piero Rinaldo at the Mayo Clinicin Rochester is assisting participants in several aspects:

> introducing generalised tandem mass spectrometry(MS/MS) testing

> standardisation of reference values for metabolicdiseases of neonates and congenital adrenalhyperplasia (CAH)

> improving overall analytical performance

> setting and sustaining the lowest achievable ratesof false-positive results

> improving and standardising confirmatory testingand short-term follow-up

Many tools are made available to evaluate and com-pare cut-off values, calculate performance, interpretresults or even share positive specimens.Training courses are also organised periodically(www.region4genetics.org/cluster1_information.aspx).Comparing our commercial methodology with thatof the QCE panels generally proved our results to bereliable.

Summary

Expanded neonatal screening is a recent metho-dology in constant evolution. Indeed, new analytesare periodically added to the aminoacid and acyl-carnitine standard panel [7, 8], meaning recurrentprocess and method validations. Consequently,permanent skills and improvement in knowledgeare required in this complex, but nevertheless fasci-nating, field.

References:

[1] Millington DS, Kodo N, Norwood DL, Roe CR. Tandem mass spectrometry:a new method for acylcarnitine profiling with potential for neonatal screeningfor inborn errors of metabolism. J Inherit Metab Dis 1990; 13: 321–4.

[2] Rashed MS, Ozand PT, Bucknall MP, Little D. Diagnosis of Inborn Errors ofMetabolism from Blood Spots by Acylcarnitines and Amino Acids ProfilingUsing Automated Electrospray Tandem Mass Spectrometry. Pediatr Res 1995;38: 324–31.

[3] Wiley V, Carpenter K, Wilcken B. Newborn Screening with Tandem MassSpectrometry: 12 Months' Experience in NSW Australia. Acta Paediatr Suppl1999; 88: 48–51.

[4] Center for Disease Control and Prevention. Using Tandem Mass Spectrometryfor Metabolic Disease Screening among Newborns. A Report of a Work Group.MMWR Recomm Rep 2001; 50: 1–34.

[5] Schulze A, Lindner M, Kohlmuller D, Olgemoller K, Mayatepek E, HoffmannGF. Expanded Newborn Screening for Inborn Errors of Metabolism by Electro-spray Ionization-Tandem Mass Spectrometry: Results, Outcome, and Impli-cations. Pediatrics 2003; 111: 1399–406.

[6] Wilcken B, Wiley V, Hammond J, Carpenter K, Screening Newborns for InbornErrors of Metabolism by Tandem Mass Spectrometry, N Engl J Med 2003; 348:2304–12.

[7] Lacey JM, Minutti CZ, Magera MJ, Tauscher AL, Casetta B, McCann M,Lymp J, Hahn SH, Rinaldo P, Matern D. Improved specificity of newbornscreening for congenital adrenal hyperplasia by second-tier steroid profilingusing tandem mass spectrometry. Clin Chem 2004; 50: 621–5.

[8] Turgeon C, Magera MJ, Allard P, Tortorelli S, Gavrilov D, Oglesbee D, RaymondK, Rinaldo P, Matern D. Combined newborn screening for succinylacetone,amino acids, and acylcarnitines in dried blood spots. Clin Chem 2008;54: 657–64.

Page 41/08LOGDIA

Newborn Screening at LiègeBoemer François, Biochemical Genetics Laboratory, Human Genetics Centre, CHU Sart-Tilman, University of Liège, Belgium

Progress Report on Diagnostics with Tandem Mass Spectrometry

Figure 1a: MRM profile of an MSUD patient.Leu/Ile: m/z = 188.1; Leu-D3: m/z = 191.1Val: m/z = 174.1; Val-D8: m/z = 182.1

Figure 1b: MRM profile of a normal patient.Leu/Ile: m/z = 188.1; Leu-D3: m/z = 191.1Val: m/z = 174.1; Val-D8 = 182.1

Psychtropic drugs are of cardinal importancefor the treatment of psychotic disorders. Bloodlevels of psychotropic drugs need to be monitoredto assure patient compliance, to reduce the risk ofintoxication due to an overdose and to rule outineffectively low levels.

Neuroleptics

Neuroleptics or antipsychotic drugs are an im-portant group of psychoactive drugs, which are cha-racterised by mood and affect inhibition. They leadto a reduction in psychomotoric activity by having acalming, relaxing and sleep-inducing effect. Theirantipsychotic effect is shown particularly in influen-cing the so-called productive psychotic symptoms,such as hallucinations, delusional, thought, andaffect disorders. An improvement in schizophrenicminus symptoms, such as apathy, loss of interest andautism, often only occurs after a longer period oftreatment. They raise stress tolerance and have antie-metic and antiallergic effects. The physical and men-tal dependency that occurs requires continuous con-trol. Clozapine, for example, can cause impairedconsciousness and cardiac symptoms (tachycardia,arrhythmia) even in the therapeutic daily dose range.

Antidepressants

The collective heading of antidepressants isunderstood as the chemically heterogeneous groupof those psychoactive drugs that have mood-activating and mood-enhancing, or anxiolytic andmood-calming effects. Among the different types,divided according to their biochemical action profile,are tricylic antidepressants (TCA), selective serotoninreuptake inhibitors (SSRI), serotonin and noradrena-lin reuptake inhibitors (SNRI), monoamine oxidaseinhibitors (MAO inhibitors), as well as noradrener-geric and specific serotonergic antidepressants(NaSSA).

Mechanisms of action

TCAs are the first choice for treating severe de-pressions. Amitryptilin has been introduced by thecompany Lundbeck as early as 1962 and this drug hasbeen the most frequently prescribed antidepressantfor many years worldwide. Antidepressants functionas inhibitors of neurotransmitters serotonin andnoradrenalin uptake in the synaptic cleft.SSRIs are not chemically related to the TCAs but they

also act in the synaptic cleft inhibiting serotoninreuptake. Compared to TCAs, SSRIs are preferredparticularly for ambulant treatment because of theirspecific receptor binding profile relating a relativelylow toxicity in case of an overdose. The newer SNRIsuch as Venlafaxin specifically inhibit serotonin andnoradrenalin reuptake.Mirtazapine is currently the only drug within theclass of noradrenergic, specifically serotonergic anti-depressants (NaSSA). As an a-antagonist it blocks thepresynaptic alpha2-autoreceptor which causes theaccumulation of noradrenalin in the synaptic cleft.Additionally. Mirtazapine has a serotonergic effect.

Routine drug monitoring

If TDM is done by HPLC, a reagent kit has tosatisfy expectations such as robustness, selectivity,sensitivity and user-friendliness. With introducingdiagnostics by LC-MS/MS which can detect coelutingsubstances, the analysis time has been significantlyreduced. The use of the MRM mode does not neces-sarily cause a reduced selectivity; actually the contra-ry is the case, selectivity increases. The MRM analysisoffers the option to select substances of interest whileomitting the detection of others in the sample. Thusthe user is not required to analyse the entire set ofdrugs within one sample. Generally detection limitsare lower with LC-MS/MS analysis than withUV/HPLC.

Since an LC-MS/MS-analysis with the MRM modewill be focussed on selected substances it will not bepossible to detect interferences. Therefore it is im-portant to use a thoroughly validated method thatensures constant analysis quality. Most of all, theanalytes need to be purified and separated from ma-trix. Matrix ingredients that have not been removedduring sample preparation have to be eliminated bymeans of an HPLC separation. This will ensure thation suppression effects are minimised or ruled outand the quantification does not suffer any compro-mises. Insufficient sample preparation and resultingion suppression might otherwise falsify results: Peaksthat should theoretically account for the sameamounts will appear different, thus distorting theresults. This effect cannot be compensated by the useof an internal standard. Further on, inadequate

sample preparation will add to a quick contamina-tion of the ion source resulting in the loss of sensitivi-ty and requiring more instrument maintenance.

Chromsystems Products

The new MassTox® analytical platform for TDMwith tandem mass spectrometry will meet all theabove-mentioned requirements. Two kits for psycho-active drugs will come on to the market as the firstproducts. One kit will cover the range of the newerantidepressants, such as SSRIs and SNRIs. A secondkit configuration will be available for analysingneuroleptics (see box for analytes). Both kits areequipped with matching 3+1 calibrators (3 levels, 1blank), and corresponding control materials (2 levels)are provided for each analyte group. Sample prepara-tion is simple and quick to carry out (see schedule),and at the same time, robust and safe. It allows prepa-ration of approx. 20 plasma or serum samples in lessthan 15 minutes. Using three internal standards,variations in individual analytes are balancedthrough sample preparation, and resilient and repro-ducible results are therefore obtained.

Further products from this range will follow.

References:

Ludewig und Regenthal, Akute Vergiftungen und Arzneimittelüberdosierungen (10.Auflage), WVG Stuttgart, 2007

Woggon, Depressionen und Psychopharmaka, pmi Verlagsgruppe Frankfurt am Main,2000

Diana Garside, Selective Serotonin Reuptake Inhibitors are Diverse Group, Clinical &Forensic Toxicology News, June 2005

Page 51/08LOGDIA

Therapeutic Drug Monitoring by Tandem Mass Spectrometry:

MassTox® Psychotropic DrugsGregor Schütze, Dr. Stephan Burghardt, Chromsystems

Neuroleptics

Analyte MRM

Aripiprazole 448,1–285

Clozapine 327,1–270

Norclozapine 313,1–270

Haloperidol 376,1–165

Olanzapine 313,1–256

Desmethylolanzapine 299,1–256

Quetiapine 384,2–253

Risperidone 411,2–191

9-OH-Risperidone 427,2–207

Antidepressants

Analyte MRM

Mirtazapine 266,1–195

SSRI

Citalopram 325,2–109

Fluoxetine 310,1–148

Desmethylfluoxetine 296,1–134

Fluovoxamine 319,2–258

Paroxetine 330,1–192

Sertraline 306,1–275

N-Desmethylsertraline 292,1–275

SNRI

Venlafaxine 278,2–260

O-Desmethylvenlafaxine 264,2–246

Duloxetine 298,1–154

Multiple Reaction Monitoring (MRM):

This MS method requires two linked mass spectrometers,which have both been statistically set to a certain mass-to-charge (m/z) ratio. The first MS selects the requiredion from ions of another nominal mass. A specificfragmentation is caused in the collision cell. The cha-racteristic daughter ion is detected in the second MS,obtaining very high selectivity.

Sample preparation:

> Mix ISTD with precipitation reagent

> Place 50 µl sample (patient sample, calibrator,control)

> Add 25 µl extraction buffer, vortex briefly and in-cubate for 2 minutes at room temperature; do not centrifuge!

> Add 250 µl precipitation reagent with ISTD, vortexfor 30 sec

> Centrifuge for 5 min at 9000 g> Dilute 100 µl supernatant with an instrument specific

amount of dilution buffer

The Chromsystems company website now offers even more service for customers.Result values of EQAS participations made with Chromsystems methods areonline now. Customers can access and print all EQAS certificates thatChromsystems has received since 2006. The online engine allows for searchingaccording to the measured parameter, issuing institute or according to theyear the assessment was made. The database is updated regularly.

A further improvement of the website is the new section on calibrators.Analogous to the quality controls section, calibrators are shown with anoverview on what is available with details being accessible through clickingon one of the products. The information comprises exemplary tables onsubstances contained and their concentrations, stability data and ordernumbers.

Page 61/08LOGDIA

Chromsystems News

Added Service:

“www.chromsystems.de“ now includes results forEQAS participations and overviews on calibrators

PRODUCT INFORMATION

MassTox® Antidepressants andNeuroleptics with Tandem-MS

> Tailormade internal standards

> Multi-level calibrators

> Minimising ion suppression effects

> Suitable for every Tandem-MS withsufficient sensitivity

The new MassTox® product group ”psychotropicdrugs by LC-MS/MS“ is extending our productbranch of diagnostics for tandem mass spectro-metry. Therapeutic drug monitoring is an optimalapplication field for mass spectrometry; a multi-tude of various substances can be measured witha single analysis in a very short time, thus ensuringa high throughput.

The new reagent kit allows for the analysis of theparent compounds as well as the most importantmetabolites. The reagents and chromatographicseparation are optimised in order to minimise ionsuppression effects and to increase the robustnessof the method. The use of multiple internal stan-dards and multi-level calibrators guarantees highprecision and accuracy of the results.

1.50 1.55 1.60 1.65 1.70 1.75 1.80 1.85 1.90 1.95 2.00 2.05199 205 212 218 225 232 238 245 251 258 265 271

Time, min

0.0

1.0e4

2.0e4

3.0e4

4.0e4

5.0e4

6.0e4

7.0e4

8.0e4

9.0e4

1.0e5

1.1e5

1.2e5

1.3e5

1.4e5

Inte

nsity

, cps

ISTD 3

ISTD 2

ISTD 1

Citalopram

Duloxetine

Fluoxetine

Desmethylfluoxetine

Fluvoxamine

Paroxetine

Sertraline

Desmethylsertraline

Venlafaxine

Desmethylvenlafaxine

1.55 1.60 1.65 1.70 1.75 1.80 1.85 1.90 1.95 2.00 2.05 2.10222 229 237 244 251 258 265 272 279 287 294 301

Time, min

0.0

5000.0

1.0e4

1.5e4

2.0e4

2.5e4

3.0e4

3.5e4

4.0e4

4.5e4

5.0e4

Inte

nsity

, cps

ISTD 2

ISTD 3ISTD 1

Aripiprazole

Clozapin

Desmethylclozapine

Haloperidole

Olanzapine

Desmethylolanzapine

Quetiapine

Risperidone

OH-Risperidone

Available soon

Fully developed symptoms of a vitamin de-ficiency, such as scurvy, pellagra and beriberi, havebecome rare in industrial countries. A sub-optimalvitamin supply seems, however, to be of im-portance in the development and course of chronicillnesses such as coronary heart disease, osteoporo-sis and cancer. There are also a number of circum-stances in which clinically significant hypovita-minosis occur that include malnutrition andundernourishment, parenteral nutrition over alonger period of time haemodialysis, malabsorp-tion, chronic alcoholism, or increased require-ments during pregnancy and lactation. A diagno-sed vitamin deficiency can be well treated; there-fore, this option should be taken into account asdifferential diagnosis.

Vitamin B1

The active form of Vitamin B1, thiamine diphos-phate (thiamine pyrophosphate, TPP), is required fora number of enzymatic reactions of carbohydratemetabolism. TPP, for example, is involved as a coen-zyme of pyruvate decarboxylase in a key step of oxi-dative glucose degradation. In particular, brain andnerve cells depend on energy from glucose; thiaminedeficiency, therefore, affects these areas in particular.

In addition, a deficit of Vitamin B1 leads to an accu-mulation of intermediary degradation productswhich have a toxic affects on musculature, myo-cardium and CNS.Pure forms of vitamin B1 deficiency are rare. It does,however, occur in alcoholics due to one-sided nutri-tion or malabsorption in the form of heart muscleweakness and psychic symptoms. In diabetics, thereare indications that vitamin B1 deficiency is involvedin vascular changes, which can lead to kidney failureand blindness in the further course of the illness. Inaddition, a Vitamin B1 deficiency can be the cause ofpolyneuropathy with dysesthesia and paralysis.

Vitamin B6

Vitamin B6 refers to the pyridoxine group pyri-doxine, pyridoxamine and pyridoxal, predecessor ofpyridoxal-5’ phosphate, which is required as coen-zyme for a number of non-oxidative reactions ofamino acid metabolism. Furthermore, it is involvedin the formation of physiologically active amines, inanabolic and catabolic metabolism changes, as wellas in the formation of red haemoglobin. Due to itsmanifold effects as coenzyme, deficiency symptomsare not always clearly discernible. In the facial region,inflammatory skin changes around the nose, eyes

and mouth are described. In babies, neuritides andspasms are prominent. As vitamin B6 is involved inthe degradation of homocysteine, any deficiencyleads to an increase in the homocysteine level, andthus indirectly raises the risk of coronary heart disease.

Analysis

The new reagent kit allows for the simultaneousdetermination of vitamin B1 in whole blood, andvitamin B6 in whole blood or plasma. Sample prepa-ration can be done with different matrices so that inthe same sequence whole blood and serum/plasmasamples can be run. In case only whole blood samplesare used, vitamins B1 and B6 can even be analysedfrom a single sample, thus sparing time and reducingcosts.

Internal Standard

The new method is distinguished by the use ofan internal standard since this is the case only withthe Chromsystems analysis. The internal standard isadded to the sample when starting the preparationthus it will undergo all measures exactly like thepatient probe. All effects that the sample preparationmay have on the result can then be seen through thevalue the internal standards produces and correctivecalculation can be made if necessary. Systematicerrors appearing due to pipetting deviations or fluctu-ation of instrumental parameters can all be compen-sated by way of calculation. The structure and con-centration of the internal standard are specificallyadapted to the Chromsystems method and furtherinterferences can be excluded. Taken together, thehighest possible precision and reproducibility aregiven.

Pre-column derivatisation

In the Chromsystems method for determiningvitamins B1 and B6 the analytes are derivatized beforethe HPLC run (pre-column derivatization). Then theprepared samples are run on an HPLC system withfluorescence detection without further modifica-tions. The required set of instrumentation is simpleand the method can be run on any HPLC-systemwith a binary pump and a programmable fluores-

Page 71/08LOGDIA

Combined Analysis without post column derivatization

Vitamins B1/B6-Analysis with Internal StandardDr. Wiebke Großberger, Chromsystems

Vitamin

Vitamin B1Thiamine

Vitamin B6Pyridoxine groupR = –CH2OHPyridoxineR = –CH2NH2PyridoxamineR = –CHOPyridoxal

Active metabolite

Thiamine diphosphate(Thiamine pyrophos-phate, TPP)

Pyridoxal-5’-phosphate(PLP)

Deficiency symptoms

BeriberiPolyneuritisPsychic changes(Wernicke and Korsakoffsyndrome)Heart muscle weakness

NeuritidesSpasmsSeborrheic dermatitisGlossitis

Structure formula

NH2

NH3C

NS

H3C

OH

N+

R

N

OH

CH3

HOH2C

Seite 81/08LOGDIA

cence detector. In contrast, a post-column derivatisa-tion needs more, at least one additional pump. Allinstruments that the liquid flow passes through haveto withstand the corrosive derivatisation reagent.Each time this reagent is pumped into the generalflow, a pulsation occurs and effects the detectorwhich causes an inconvenient signal to noise ratio.Depending on the length of the reactor (capillarywhere the derivatisation reaction occurs) peaks canbroaden reducing the result quality.Chromsystems has chosen the precolumn derivatisa-tion to rule out all of these disadvantages.

Summary

Using the new Chromsystems reagent kit, thephysiologically active forms of vitamins B1 and B6 –thiamine pyrophosphate and pyridoxal-5’ phosphate– can now be simultaneously determined. The me-thod enables the processing of different matrices, sothat serum, plasma and whole blood samples can beused in the same sample preparation. If whole bloodfor both vitamins is used, both can be analysed froma single sample. The analytes are derivatised beforethe HPLC run in the course of sample preparation.

Separation occurs by means of switching betweentwo different mobile phases on the same column andsubsequent fluorescence detection with two differentwavelengths.A further plus of the Chromsystems method is theuse of an internal standard, enabling the compensa-tion of losses during sample preparation, or inaccura-cies during injection.Matching quality controls and a calibrator guaranteeprecise and reliable quantification.

Chromsystems News

Chromsystems increases production capacitiesand invests in research and development

Chromsystems reacts to the increased demandfor its products worldwide by investing into newfacilities and new qualified employees. The newcapacities lay the foundation for further growth ofthe company.

New tandem mass spectrometers and new personnelfavours the expansion process and accelerate thedevelopment of future products. We are growingwhile maintaining constant quality.

The production department has been extended intonew rooms and new instrumentation support aneven higher level of automation; additionally con-sidering the new employees, Chromsystems’ produc-tion capacities have thus multiplied. Customers inmore than 75 countries can now be provided fasterwith more products.

Naturally increased performance is also implementedat other departments such as customer services,logistics and quality management. From developmentto delivery customers are backed by even moreChromsystems service.

Within three years the number of Chromsystemstests used worldwide has doubled. Our infrastructureis growing in parallel.

PRODUCT INFORMATIONNEW:Combined Analysisof Vitamins B1 and B6

The new reagent kit allows for the combinedanalysis of vitamin B1 in whole blood and vitaminB6 in whole blood, plasma and serum. The samplepreparation has the capacity to process differentmatrices, thus one analystic sequence can includewhole blood sample as well as plasma or serumsamples. The necessary derivatisation of thevitamin molecules is performed during the samplepreparation which renders the common post-column derivatisation unnecessary. The separationis done with two separate mobile phases on oneHPLC column with fluorescence detection at two

different wavelengths. An internal standard andmatching quality controls ensure precise and safequantifications.

Ordering information:52000 Reagent kit0164 Vitamins B1/B6 Whole Blood Control, Bi-Level (I+II)0165 Vitamins B1/B6 Whole Blood Control, Level I0167 Vitamins B1/B6 Whole Blood Control, Level II0031 Vitamin B6 Plasma Control, Bi-Level (I+II)0038 Vitamin B6 Plasma Control, Level I0039 Vitamin B6 Plasma Control, Level II

> Analysis optionally in whole blood or plasma

> Both vitamins in one samplepreparation and one run

> No post-column derivatisation> With internal standard

mV

Minutes

Vit. B

6

Vit. B

1

Int.

Stand

ard

-5,00

0,00

5,00

10,00

15,00

20,00

25,00

30,00

35,00

40,00

45,00

50,00

1,00 2,00 3,00 4,00 5,00 6,00 7,00

Bone, a highly differentiated hard connectivetissue, is involved in all movements of the bodyand is largely composed of a calcium phosphatecomplex. The latter accounts for approximately70 % of bone material and lends it its firmness.The basis of bone is formed by type I collagenconnective tissue fibres (20 % content) that areresponsible for the elasticity of bones. The firmstructure of the collagen network is achieved bycross-linking (pyridinium crosslinks: pyridinoline,PYD and deoxypyridinoline, DPD) (Fig. 1). Thepyridinium crosslinks are derivatives of the basicamino acid lysine.

Bone metabolism – an equilibrum

Bone is subject to continuous trans-formation, with the result that the humanskeleton is renewed multiple times in thecourse of a lifetime. This takes place throughthe activity of osteoblasts (synthesis), alsoknown as osteocytes in their more diffe-rentiated form, and osteoclasts (breakdown),the cellular components of bone. Synthesisand breakdown are parallel processes, whichusually take place in mutual equilibrium.This results in a relatively stable bonedensity which can be further supported bysports and a calcium-rich, healthy diet.

Degradation with increasing age

Peak bone mass is generated untilapproximately 20 years of age, giving wayto an annual 0.5–1.5 % decline thereafter.During this inevitable decline, a shift inthe synthesis-breakdown balance may resultin abnormal loss of bone mass. A high rateof breakdown, which may be due to avariety of reasons, promotes the develop-ment of osteoporosis, which renders theskeletal system – the vertebrae, pelvis and femoralneck bones being at a particularly increased risk offracture – porous and friable.

Osteoporosis

Osteoporosis accounts for about 2 millionfemoral neck fractures annually. The associated costsof treatment constitute a significant economic bur-

den. The World Health Organisation (WHO) recogni-zed this trend and listed osteoporosis among the top10 diseases. In Germany, the direct and indirect costsof osteoporosis amount to approximately 2.5–3billion Euro annually. At a rough estimate, the diseaseaffects 8–10 % of the German population. At least75 % of osteoporosis sufferers are female. Numerousscientific studies show that the risk of developingosteoporosis is highest for postmenopausal women,men with testosterone depletion, and the over-70population. This disease of the skeletal system ismarked by a disproportionately high decline in themineral (hydroxyl apatite) and organic (osteoid) partof bone, and should not be confused with normalage-related bone loss.

Therapy

As there are few ways of increasing bone mass,contemporary therapeutic measures are mainly basedon preserving bone density, which can be achievedamong other things by treatment with bisphospho-nates, calcium and calcitonin, or by estrogen therapyin the postmenopausal female population. It isimportant to note in this context that calcium, an

essential factor in bone metabolism, is absorbed inthe small intestine and ultimately into the bonesonly in the presence of vitamin D. However, vita-min D supply is inadequate in many cases. An esti-mated approximately 50 % of the population in theGermany-Austria-Switzerland area have a vitamin Ddeficiency, indirectly and ultimately resulting indeterioration of their bone density status. Osteo-porosis must be treated as a disease of multifactorialetiology whose outcome is positively modifiable ifthe condition is diagnosed in good time.

Diagnosis

Due to the fact that bone mass losses are identi-fiable by osteodensitometry (bone densitymonitoring) only at a very late stage, thereis a need to identify significant bone lossbeforehand in laboratory tests as a basisfor a targeted response. The best markerfor identifying above-average bone loss isassay of urinary pyridinoline crosslinks, asthe latter are almost entirely from bone,constitute a non-dietary marker, and corre-late directly with the severity of osteopo-rosis. Increased crosslink clearance mayalso point to other bone degenerationdisorders, including hyperthyroidism, bonemetastasis, Paget's disease, osteoarthritis,osteomalacia, rheumatoid arthritis andcancer-related hypercalcemia.

Chromsystems Analysis

The most reliable laboratory methodfor evaluating increased bone collagen losswas established by Chromsystems as partof their osteoporosis diagnostics range andis now in routine use, along with assay ofvitamin D status with the Chromsystemsreagent kit (DIALOG 2/2007; page 1ff.).

The method's high sensitivity enables determinationof the total pyridinium crosslinks content in theform of pyridinoline (PYD) and deoxypyridinoline(DPD), and also permits assay of the free pyridinolineconcentration. This is done by highly specific samplepreparation during which interfering fluorophorsare removed by solid phase extraction. The resultanteluates are then analyzed by isochromatic HPLC (Fig.2). A hydrolysis-proof internal standard rounds ofthis precise and stable diagnostic lab assay system.

Page 91/08LOGDIA

Clinical relevance of pyridinium crosslinksDr. Richard Lukacin, Chromsystems

Marker of increased bone loss

PRODUCT INFORMATION

> Hydrolysis resistant internal standard> Easy sample preparation> Accurate and reliable calibration

Pyrid

inoli

ne

Deox

ypyr

idino

line

Int.

Stand

ard

Minutes

This reagent kit allows for the simple and reliablequantification of the total amount of the twourinary Crosslinks, Pyridinoline (PYD) and Deoxy-pyridinoline (DPD) with an isocratic HPLC system.The use of an acidic hydrolysis resistant InternalStandard (IS) compensates losses and variationin the extraction procedure and increases precision

and accuracy. After extraction on Sample CleanUpColumns as well as several washing steps inorder to remove interferingsubstances, PYD, DPD,and IS are eluted. The chromatographic separationis carried out on an RP column followed byfluorescence detection.

Crosslinks in Urine

Ordering information:48000 Reagenzienkit0045 Crosslinks Urine Control, Bi-Level (I +II)0046 Crosslinks Urine Control, Level I0047 Crosslinks Urine Control, Level II

Figure 1

Desoxypyridinoline (Dpd)NH2

NH2

HOOC COOH

HO3

+N

NH2

HOOC

Pyridinoline (Pyd)NH2

NH2

HOOC COOH

HO3

+N

NH2

HOOC

OH

The C.H.R. de la Citadelle Regional Hospital inLiege is a general hospital with 1036 beds coveringall disciplines of stationary and ambulant medicalcare as well as emergency services from neonato-logy to geriatrics. It also encompasses certain uni-versity clinical services such as neurology, paedia-trics etc. Each year the hospital racks up about300,000 inpatient days and more than 400,000outpatients’ appointments. With about 200 admis-sions every 24 hours, its A & E department is amongthe largest in Belgium.

The Clinical Biology Laboratory deals with about1250 cases/biological assessments every 24 hours.More than 4,000,000 tests are carried out per year.In fact, it serves not only the hospital itself but alsothe geriatric and psychiatric hospitals of the Liegeregion and operates as a subcontract laboratory forother clinical biology laboratories.

The Toxicology, TDM and Endocrinology Laboratoryhas the status of a specialist unit working alongsidethe chemistry/haematology/haemostasis unit, whichin turn operates 24 hours per day. It is equippedwith extensive analytical facilities encompassingHPLC/DAD, HPLC/Fluorescence and Electrochemi-stry, HPLC/MS/MS and GC/MS with the option of“Headspace Sampling”. It also has automated equip-ment for ASPEC™ solid phase extraction procedures.

The “TDM” or Therapeutic Drug Monitoring area isbroken down into two levels:

The first level is carried out by the chemistry/haematology/haemostasis technical unit as an emer-gency service for all procedures that can be carriedout using immunology kits.Thus, the laboratory provides a whole range of testsincluding those for “conventional” anti-epileptics(phenobarbital, phenytoin, carbamazepine and val-proate), cardiotonics, antibiotics, caffeine, lithiumetc. as well as the first quick tests for benzodiazepines,tricyclic antidepressants etc.

The second level is carried out by the ToxicologyLaboratory itself and includes unconventional anti-epileptics, benzodiazepines, antidepressants, neuro-leptics, anti-arrythmics, anti-HIV medication etc.

In order to achieve this, the Toxicology Laboratorysuccessfully uses two types of methods, those de-veloped and validated in-house and the followingChromsystems kits:

> Benzodiazepines and Antidepressants

> Antiepileptics in high-resolution mode andlevetiracetam

> Amiodarone and desethyl metabolite

> Neuroleptics (Olanzapine and Clozapine)

> Anti-HIV

> Itraconazole

For certain applications, we have modified theChromsystems kits so as to offer a better responseto our local demands. Clearly this required thevalidation of new parameters in our laboratory.Our quality system prompted us to verify certainanalytical parameters out of the Chromsystems kitssuch as the detection/quantification limit, linearitylimits etc.

The laboratory received its ISO 9001:2000 certificationin February 2008. The traceability of all constituentelements of the kits (HPLC column, standards, con-trols etc) was a key element in the specific qualityapproach of the Toxicology Laboratory.

Need for Therapeutic Drug Monitoring

Therapeutic Drug Monitoring finds answers tothe following questions:

• What is the active mechanism of the medication?• What relationship is there between Pharmacokinetics/

Pharmacodynamics relationship? (dose/concentration/effect)

• How long should the drug be administered?• Is there any intra- and/or inter-individual PK varia-

bility?• Is there any potential for interaction between medi-

cations?• What is the reference range of the drug?

To the limited extent to which we can respond tosome, if not all of these questions, it is useful tocarry out the Therapeutic Drug Monitoring of themedication concerned.

Antiepileptics respond to these criteria particularlywell, even though we should draw attention to thefact that the relevance of blood concentration doesvary depending on the molecule (c.f. recommendedliterature)

Figure 1 shows an example of the blood testing oflamotrigine and oxcarbazepine with its active meta-bolite, 10-OH-carbazepine with the assistance of theantiepileptics kit used in high resolution mode.

Acknowledgements:

The authors would like to thank the technicalstaff at the Toxicology Laboratory, namely Ms Berna-dette Lepage, Ms Christine Paquet, Ms Marie-LuceStassart, Mr Jean-Paul Joris and Mr Serge Mellen.

Literature:

http://www.iatdmct.org, Internetseite der “International Association of TherapeuticDrug Monitoring and Clinical Toxicology“

Drug monitoring data pocket guide II. Washington, DC: AACC Press, 1994

Evans WE, Oellerich M (Hrsg.): Therapeutic drug monitoring clinical guide. U.S.:Abbott Laboratories Abbott Park, IL: 1984

Johannessen SI et al.,Therapeutic drug monitoring of the newer antiepilepticdrugs.Ther Drug Monit. 2003 Jun;25(3):347-63.

Neels HM et al. Therapeutic drug monitoring of old and newer anti-epilepticdrugs. Clin Chem Lab Med. 2004;42(11):1228-55.

Suivi thérapeutique pharmacologique pour l’adaptation des médicaments. Coord.P. Marquet. Ed. Elsevier. Coll. Option-Bio. 2004 :539pp

Therapeutic Drug Monitoring and Clinical Biochemistry. N. Capps. AACC Press1997. 178 Seiten

Warner A Annesley T (Hrsg.): Guidelines for therapeutic drug monitoring services1999. 110 Seiten. National Academy of Clinical Biochemistry Washington, DC.

Wu AHB, Broussard LA, Hoffman RS, Kwong TC, McKay C, Moyer TP et al. (Hrsg.):Laboratory medicine practice guidelines: recommendations for the use of laboratorytests to support the impaired and overdosed patient from the emergency department[Draft Guidelines]. Washington, DC: National Academy of Clinical Biochemistry(http://www.nacb.org/Toxicology_LMPG.stm).

Page 101/08LOGDIA

TDM in a regional hospital:C.H.R. de la Citadelle in LiègeThierry Gougnard, Toxicology Laboratory, TDM and Endocrinology / Jean-Marc Minon, Clinical Biology Laboratory

Lam

otro

gine

10-O

H-C

arba

maz

epin

e

Oxc

arba

maz

epin

e

Int.

Sta

ndar

d

Figure 1

Page 111/08LOGDIA

Proven Performance:

Solutions for Demanding Clinical Analysis atApplied Biosystems/MDS Analytical TechnologiesLisa Sapp, Product Manager Applied Biosystems/MDS Analytical IndustriesApplied Biosystems/MDS Analytical Technologies is a joint venture between Applera Corporation and MDS Inc.

Clinical research laboratories around the worldare discovering the analytical power of LC-MS/MSand its ability to accurately identify and quantifyminute amounts of compounds in complex samplematrices such as blood, urine and oral fluid.Innovative LC-MS/MS methods are being used inplace of conventional analysis to study inbornerrors of metabolism and metabolic profiles andto identify therapeutic drugs, drugs of abuse, diseasemarkers, and toxic compounds.

Where conventional techniques such as GC/MS,LC/UV or immunoassays often lack the requiredspecificity, sensitivity or robustness, more and moreclinical researchers are looking to LC-MS/MS. Hereare some reasons:

> The Right Answer, the First TimeSuperior sensitivity and low detection limits combinedwith high specificity lead to better data and moreconfidence in your results.

> Faster Time to ResultLC-MS/MS significantly reduces sample preparationand LC run times and increases throughput dramatically.You get results in minutes, not hours.

> Ease of UseLC-MS/MS methods can be implemented quickly andeasily, even in laboratories without extensive massspectrometry experience.

> Broader Application CoverageThe flexibility and versatility of LC-MS/MS detectionincrease the type and variety of research you can performwith a single instrument.

> Easy Analyte UpdateWith LC-MS/MS you can modify methods easily andincorporate new analytes quickly, without extensiveredevelopment.

> More Compounds Per Analysis,More Analysis Per DayFast analysis times and simultaneous screening of bothknown and unknown compounds in the same run cantake your throughput to an entirely new level.

> Maximum Instrument UptimeRugged, robust LC-MS/MS methods and instrumentationmaximize instrument uptime and allow you to runthousands of samples with minimal downtime.

Applied Biosystems/MDS Analytical Technologies isthe No.1 LC-MS/MS supplier with over 12,000 in-struments shipped to customers around the world. The company has 13 global customer support labswith more than 170 global application scientists andover 300 global service support engineers. The globalnetwork serves more than 30,000 life science labora-tories in over 150 countries, offering proven techno-logy with expert technical support and service.

Whether you need a cost-effective solution for inve-stigating high-throughput therapeutic drug monito-ring, the utmost in accuracy and sensitivity to studypanels of steroids, or the speed and versatility toidentify and quantify hundreds of targeted drugssimultaneously, Applied Biosystems/MDS AnalyticalTechnologies has an LC-MS/MS solution to meetyour requirements. We offer the widest range of LC-MS/MS systems to address existing and new applica-tion areas. These include the entry-level API 2000™,the API 3200™, the API 4000™ LC-MS/MS Systemsand the gold-standard API 5000™, which offersindustry leading sensitivity. Applied Biosystems/MDSAnalytical Technologies LC-MS/MS systems are perfectfor routine, research and academic applications thatrequire the ultimate performance and reliability.

As our business continues to grow we are alwayslooking for new opportunities to meet customer

needs by making our products easier to use andimprove customer productivity.

In line with this philosophy, Applied Biosystems isworking with Chromsystems to provide a completesolution to study inborn errors of metabolism.Chromsystems manufactures reagent kits, qualitycontrols and calibrators to provide clinical laboratoriesworldwide with a solution for fast and easy samplepreparation based on complete reagent kits contai-ning all material and know-how for a reliable, repro-ducible analysis rendering self made methods obso-lete. The solution will include Chromsystems Kitsto measure Amino Acids and Acylcarnitines, an LC-MS/MS system from Applied Biosystems/MDS Ana-lytical Technologies and Cliquid® Software methodscustomized for the Chromsystems Amino Acid andAcylcarnitine Kits.

Cliquid® Software was developed by Applied Biosys-tems/MDS Analytical Technologies and representsan entirely new approach to mass spectrometrysoftware. It was designed to improve data qualityand throughput through reduced training, moreautomation and better workflow control. With itssecure user login, simplified four step workflow,preconfigured methods, data analysis and reportingtools Cliquid® Software has been designed with theclinical research lab in mind.As the world leader in LC-MS/MS, Applied Biosys-tems/MDS Analytical Technologies is dedicated tothe development of advanced mass spectrometrytechnology that has been widely accepted as a flexible,efficient and cost effective clinical research tool.

http://info.appliedbiosystems.com/clinicalresearch

For Research Use Only. Not for use in diagnostic procedures.Applera, Applied Biosystems and AB (design) are registered trademarks of Applera Corporationor its subsidiaries in the US and/or certain other countries. API 2000, API 3200, API 4000and API 5000 are trademarks and Cliquid is a registered trademark of Applied Biosystems/MDSAnalytical Technologies, a joint venture between Applera Corporation and MDS Inc. All othertrademarks are the sole property of their respective owners.© 2008 Applera Corporation and MDS Inc. Joint Owners. All rights reserved.

API 4000™ LC-MS/MS SystemAPI 3200™ LC-MS/MS SystemAPI 2000™ LC-MS/MS System

In the last years technical innovations in analysis by mass spectrometry have generatednew possibilities for in vitro diagnostics. This method, originally complex anddemanding, is now supported by user-friendly and precise instruments which qualifymass spectrometry for routine diagnostics. Implementing this technologic trend,Chromsystems is investing into the development of diagnostic kits, quality controlsand calibrators for tandem mass spectrometry.Basically Chromsystems products can be run with all instruments of current producers.To ensure this compatibility Chromsystems R&D is carried out on different instrumentsfrom suppliers such as Applied Biosystems, Thermo Scientific and Waters Corporation.

Chromsystems products yield reliable and reproducible results on all mass spectrometerswith sufficient sensitivity. Currently, Chromsystems has started a cooperation withApplied Biosystems/MDS Analytical Technologies (AB). Within this frame relationship,AB offers mass spectrometers with the required quality and respective instrumentservice to Chromsystems customers. AB also supports their clinical diagnostic researchcustomers with reagent kits and quality control material from Chromsystems.The following article by Lisa Sapp, product manager in charge at Applied Biosystems,gives an overview on the current mass spectrometers and accompanying software(The Editor).

Page 121/08LOGDIA

Imprint

Publisher:ChromsystemsInstruments & Chemicals GmbHHeimburgstrasse 381243 Munich, Germany

Phone: +49 89 18930-200Fax: +49 89 18930-299E-mail: mailbox@chromsystems.de

Editor:Gabriel Erlenfeld

Design:Lengnick Print- & Media Design

Print:Stulz-Druck & Medien, München

Edition July 2008

Dates

Chromsystems will be represented 2008at the following national and internatio-nal fairs:

> 29–31 July 2008AACC, Washington DC

> 02–05 September 2008SSIEM Soc Stud Inborn Errors Met, Lissabon

> 04–05 September 2008GTFCh-Workshop, Basel

> 08–11 September 2008Journées Fr. Spectromét. d. Masse, Grenoble

> 16–19 September 2008Swiss MedLab-Int. Congress, Montreux

> 21–24 September 2008DGKL-Jahrestagung, Mannheim

> 24–27 September 2008AGATE-TDM Symposium, Regensburg

> 25th September 2008HPLC-Tagung, Würzburg

> 26–27 September 2008TDM Conference, Warschau

> 28–02 Sept./Oct. 2008IFCC Worldlab 2008, Fortaleza

> 28–31 October 2008SIBIOC, Rimini

> 11–14 November 2008LC-MS/MS Meeting, Montreux

> 19–22 November 2008MEDICA, Düsseldorf

Chromsystems Training

New workshops for diagnosticsby LC-MS/MS

Routine diagnostics byTandem Mass Spectrometry

Collect knowledge and experience – and shareit. This is the Chromsystems attitude in clinicalroutine diagnostics. Our products are not the onlytarget we put our expertise into. The Chromsystemsworkshops and seminars are excellent opportunitiesto acquire or broaden knowledge and experienceand a good condition for rapid and reliable dia-gnostic results.

Now Chromsystems is a competent partner forroutine diagnostics by tandem mass spectrometry,too. Our products are developed to be applicableon all mass spectrometers with sufficient sensitivity,thus they can be run on all tandem mass spectro-meters currently available. Accordingly, the Chrom-systems laboratories are furnished with equipmentfrom all major MS instrumentation producers.

LC-MS/MS workshops

Contrary to common belief presuming thatthe specificity of mass spectrometry analyses ren-ders sample preparation unnecessary, our R & Defforts have shown the requirement of it. In order

to benefit from the outstanding qualities of MSdiagnostics, it is crucial to rule out matrix interfe-rences. Beyond that, MS instrumentation stillbeing demanding, it is necessary to adapt andequilibrate the mass spectrometer for the respectiveanalysis in order to produce reliable results in dailyroutine.

The new mass spectrometry workshops prepareattendants for correct patient sample collection,sample preparation and instrument use in theroutine diagnostic setting. Background informationis given during lectures in the morning while inthe afternoon attendants are trained during practi-cal courses in particular workshop laboratories.Facilities offer exercise on tandem mass spectro-meters from leading manufacturers such as AppliedBiosystems, Thermo Scientific and Waters Corpo-ration. One focus besides sample treatment isoptimising instrument parameters for diagnosticuse. Another focus is troubleshooting and evalua-tion of data.Attendees passing this workshop have an in-depthoverview on the diagnostic parameters of interest,on how to yield correct results and how to interpretthese. Attendance is limited to 15 participants.Practical courses are performed in small groupswith selected highlights.