Name: multiplex therapeutic drug monitoring by isotope-dilution hplc-ms/ms of antibiotics in critical illnesses
Uploaded: 2018-08-30T15:00:00
Description: Watch this Scientific Journal Video about Multiplex Therapeutic Drug Monitoring by Isotope-dilution HPLC-MS/MS of Antibiotics in Critical Illnesses at JoVE.com

Författarna tacka Dr Schütze för hans hjälp med att upprätta den presenterade metoden och Dr Zoller för värdefull input om korrekt kalibreringsområdet. Författarna också erkänna den tekniska personalen i anläggningen masspektrometri.

Obs: Det rekommenderas att arbeta i dragskåp vid hantering av organiskt lösningsmedel, såsom metanol. Förbereda alla buffertar och mobila faser i mätkolvar. Om inte annat anges, kan lösningarna förvaras vid rumstemperatur i upp till 1 månad efter beredning.
1. beredning av kalibratorer och kvalitetskontroll prover
Obs: En motsvarande analys datablad för beredning av lager och spike lösningar ges i den Kompletterande fil. På grund av spårba...

<ol>
	<li>Fleischmann, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+Global+Incidence+and+Mortality+of+Hospital-treated+Sepsis.+Current+Estimates+and+Limitations.">Assessment of Global Incidence and Mortality of Hospital-treated Sepsis. Current Estimates and Limitations.</a> American Journal of Respiratory and Critical Care Medicine. 193 (3), 259-272 (2016).</li><li>Dellinger, R. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surviving+Sepsis+Campaign:+international+guidelines+for+management+of+severe+sepsis+and+septic+shock,+2012.">Surviving Sepsis Campaign: international guidelines for management of severe sepsis and septic shock, 2012.</a> Intensive Care Medicine. 39 (2), 165-228 (2013).</li><li>Lodise, T. P., Drusano, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacokinetics+and+pharmacodynamics:+optimal+antimicrobial+therapy+in+the+intensive+care+unit.">Pharmacokinetics and pharmacodynamics: optimal antimicrobial therapy in the intensive care unit.</a> Critical Care Clinics. 27 (1), 1-18 (2011).</li><li>Macedo, R. S., Onita, J. H., Wille, M. P., Furtado, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacokinetics+and+pharmacodynamics+of+antimicrobial+drugs+in+intensive+care+unit+patients.">Pharmacokinetics and pharmacodynamics of antimicrobial drugs in intensive care unit patients.</a> Shock. 39, 24-28 (2013).</li><li>Petersson, J., Giske, C. G., Eliasson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standard+dosing+of+piperacillin+and+meropenem+fail+to+achieve+adequate+plasma+concentrations+in+ICU+patients.">Standard dosing of piperacillin and meropenem fail to achieve adequate plasma concentrations in ICU patients.</a> Acta Anaesthesiologica Scandinavica. 60 (10), 1425-1436 (2016).</li><li>Abdul-Aziz, M. H., Lipman, J., Mouton, J. W., Hope, W. W., Roberts, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applying+pharmacokinetic/pharmacodynamic+principles+in+critically+ill+patients:+optimizing+efficacy+and+reducing+resistance+development.">Applying pharmacokinetic/pharmacodynamic principles in critically ill patients: optimizing efficacy and reducing resistance development.</a> Seminars in Respiratory and Critical Care Medicine. 36 (1), 136-153 (2015).</li><li>Imani, S., Buscher, H., Marriott, D., Gentili, S., Sandaradura, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Too+much+of+a+good+thing:+a+retrospective+study+of+beta-lactam+concentration-toxicity+relationships.">Too much of a good thing: a retrospective study of beta-lactam concentration-toxicity relationships.</a> Journal of Antimicrobial Chemotherapy. 72 (10), 2891-2897 (2017).</li><li>Ventola, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+antibiotic+resistance+crisis:+part+1:+causes+and+threats.">The antibiotic resistance crisis: part 1: causes and threats.</a> Pharmacy &amp; Therapeutics. 40 (4), 277-283 (2015).</li><li>Pulcini, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibiotic+stewardship:+update+and+perspectives.">Antibiotic stewardship: update and perspectives.</a> Clinical Microbiology and Infection. 23 (11), 791-792 (2017).</li><li>Cairns, K. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+a+multidisciplinary+antimicrobial+stewardship+team+on+the+timeliness+of+antimicrobial+therapy+in+patients+with+positive+blood+cultures:+a+randomized+controlled+trial.">The impact of a multidisciplinary antimicrobial stewardship team on the timeliness of antimicrobial therapy in patients with positive blood cultures: a randomized controlled trial.</a> Journal of Antimicrobial Chemotherapy. 71 (11), 3276-3283 (2016).</li><li>Baur, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+antibiotic+stewardship+on+the+incidence+of+infection+and+colonisation+with+antibiotic-resistant+bacteria+and+Clostridium+difficile+infection:+a+systematic+review+and+meta-analysis.">Effect of antibiotic stewardship on the incidence of infection and colonisation with antibiotic-resistant bacteria and Clostridium difficile infection: a systematic review and meta-analysis.</a> Lancet Infectious Diseases. 17 (9), 990-1001 (2017).</li><li>Roberts, J. A., Norris, R., Paterson, D. L., Martin, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+drug+monitoring+of+antimicrobials.">Therapeutic drug monitoring of antimicrobials.</a> British Journal of Clinical Pharmacology. 73 (1), 27-36 (2012).</li><li>Paal, M., Zoller, M., Schuster, C., Vogeser, M., Schutze, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+quantification+of+cefepime,+meropenem,+ciprofloxacin,+moxifloxacin,+linezolid+and+piperacillin+in+human+serum+using+an+isotope-dilution+HPLC-MS/MS+method.">Simultaneous quantification of cefepime, meropenem, ciprofloxacin, moxifloxacin, linezolid and piperacillin in human serum using an isotope-dilution HPLC-MS/MS method.</a> Journal of Pharmaceutical and Biomedical Analysis. 152, 102-110 (2018).</li><li>Vogeser, M., Seger, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pitfalls+associated+with+the+use+of+liquid+chromatography-tandem+mass+spectrometry+in+the+clinical+laboratory.">Pitfalls associated with the use of liquid chromatography-tandem mass spectrometry in the clinical laboratory.</a> Clinical Chemistry. 56 (8), 1234-1244 (2010).</li><li>United States Pharmacopeia and National Formulary. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+&#60;621&#62;.">Chapter &#60;621&#62;.</a> CHROMATOGRAPHY (USP 37-NF 32 S1). , 6376-6385 (2014).</li><li>Wong, G., Sime, F. B., Lipman, J., Roberts, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+do+we+use+therapeutic+drug+monitoring+to+improve+outcomes+from+severe+infections+in+critically+ill+patients?.">How do we use therapeutic drug monitoring to improve outcomes from severe infections in critically ill patients?.</a> BMC Infectious Diseases. 14, 288 (2014).</li><li>. Cefepime hydrochloride: Highlights of prescribing information Available from: <a target="_blank" href="https://www.accessdata.fda.gov/drugsatfda_docs/Label/2016/050679s040lbl.pdf">https://www.accessdata.fda.gov/drugsatfda_docs/Label/2016/050679s040lbl.pdf</a> (2016)</li><li>. Meropenem: Highlights of prescribing information Available from: <a target="_blank" href="https://www.accessdata.fda.gov/drugsatfda_docs/Label/2008/050706s022lbl.pdf">https://www.accessdata.fda.gov/drugsatfda_docs/Label/2008/050706s022lbl.pdf</a> (2006)</li><li>. Ciprofloxacin hydrochloride: Highlights of prescribing information Available from: <a target="_blank" href="https://www.accessdata.fda.gov/drugsatfda_docs/Label/2016/019537s086lbl.pdf">https://www.accessdata.fda.gov/drugsatfda_docs/Label/2016/019537s086lbl.pdf</a> (2016)</li><li>. Moxifloxacin hydrochloride: Highlights of prescribing information Available from: <a target="_blank" href="https://www.accessdata.fda.gov/drugsatfda_docs/Label/2010/021277s038lbl.pdf">https://www.accessdata.fda.gov/drugsatfda_docs/Label/2010/021277s038lbl.pdf</a> (2010)</li><li>. Linezolid: Highlights of prescribing information Available from: <a target="_blank" href="https://www.accessdata.fda.gov/drugsatfda_docs/Label/2012/021130s028lbl.pdf">https://www.accessdata.fda.gov/drugsatfda_docs/Label/2012/021130s028lbl.pdf</a> (2011)</li><li>. Piperacillin and Tazobactam: Highlighs of prescribing information Available from: <a target="_blank" href="https://www.accessdata.fda.gov/drugsatfda_docs/Label/2017/050684s88s89s90_050750s37s38s39lbl.pdf">https://www.accessdata.fda.gov/drugsatfda_docs/Label/2017/050684s88s89s90_050750s37s38s39lbl.pdf</a> (2017)</li><li>Zander, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+biobanking+conditions+on+six+antibiotic+substances+in+human+serum+assessed+by+a+novel+evaluation+protocol.">Effects of biobanking conditions on six antibiotic substances in human serum assessed by a novel evaluation protocol.</a> Clinical Chemistry and Laboratory. 54 (2), 265-274 (2016).</li><li>Zander, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+piperacillin,+tazobactam,+cefepime,+meropenem,+ciprofloxacin+and+linezolid+in+serum+using+an+isotope+dilution+UHPLC-MS/MS+method+with+semi-automated+sample+preparation.">Quantification of piperacillin, tazobactam, cefepime, meropenem, ciprofloxacin and linezolid in serum using an isotope dilution UHPLC-MS/MS method with semi-automated sample preparation.</a> Clinical Chemistry and Laboratory. 53 (5), 781-791 (2015).</li></ol>

I detta manuskript, redovisar vi protokollet för en enkel och robust tandem mass spectrometry-baserad metod för kvantifiering av ofta använda antibiotika i ICU19, nämligen cefepime, meropenem, ciprofloxacin, moxifloxacin, linezolid, och piperacillin14. Ett kalkylblad medföljer manuskriptet för beredning av antibiotika stamlösningar, kalibratorer och kvalitetskontroller, med hänsyn till renheten av antibiotika och molekylvikten för deras counterions. Med tanke på a...

Även om antibiotika har revolutionerat praktisk medicin, förbli svåra bakterieinfektioner en ledande orsak till sjuklighet och dödlighet i kritiska sjukdomar1. I detta avseende är snabb administrering av en lämplig anti-infective i en adekvat dosering av översta betydelse för sjukdom kontroll2.
En växande mängd bevis visar att empirisk behandling med bredspektrumantibiotika blir alltmer problematisk med komplexiteten i patientpopulationer. Detta är särskilt sant för intensivvårdsavdelningar (IVA), där en enorm interindividuell variabilitet av viktiga farmakokinetiska (PK) parametrar observeras ofta3,4. Följaktligen, ICU patienter löper överhängande risk för sub terapeutiska nivåer med faran av en otillräcklig terapeutisk framgång5,6. Då igen, utsätts patienter onödigt för alltför höga antibiotiska koncentrationerna som kan resultera i allvarliga biverkningar med inga kliniska fördelar7. Både antibiotika missbruk och otillräcklig dosering har också underblåst spridning av antibiotikaresistens, som blir ett växande hot mot folkhälsan8.
Att förbättra användningen av antibiotika och att bevara deras effectivenessas har länge som möjligt, Världshälsoorganisationen lanserat en global handlingsplan om antibiotikaresistens i 20159. Antibiotikum stewardship program utgör en viktig hörnsten i återhållsam antimikrobiella användning i nationella folkhälsan strategier10, att hjälpa klinikerna att avsevärt förbättra kvaliteten på patientvården11 och, samtidigt, att minska antibiotikaresistens12. Antimikrobiella dosering hos enskilda patienter genom tillämpning av terapeutisk övervakning (TDM) är ett viktigt instrument i detta sammanhang13.
Hittills, kommersiellt tillgängliga TDM finns analyser endast tillgängliga för glykopeptider antibiotika och aminoglykosider. Kvantifiering av ämnen från andra klasser kräver vanligen en egen metodutveckling eller validering som kan vara besvärligt. Vi presenterar därför i detalj protokollet för en robust mass spectrometry-baserad analys som kan användas för kvantifiering av de mest relevanta antibiotika i ICU inom deras kliniska relevanta koncentration spänner14. Metoden nyligen etablerats i vår masspektrometri anläggning och har tillämpats för rutinen TDM i ICU sedan dess. Proceduren använder en okomplicerad och enkel analytisk inställning med en enhetlig prov rensning, vilket möjliggör ett snabbt genomförande av antibiotika TDM i den utmärkta servicen och de suveräna faciliteterna med masspektrometri kapacitet.
Protokollet beskrivs här var optimerad för kvantifiering av cefepime, meropenem, ciprofloxacin, moxifloxacin, linezolid och piperacillin i humant serum med isotopen utspädning vätskekromatografi (LC) i kombination med en tandem mass masspektrometri (MS/MS). För Isotoputspädning LC-MS/MS metod läggs stabil isotop-märkt föreningar till ett urval av intresse med en specifik matris (t.ex., serum). Isotop-märkt standarder kan skiljas från sin omärkta motsvarighet, nämligen analyten av intresse, på grund av olika molekylvikter av naturliga molekylen och fragmentering produkter, kallas en förälder-ion-till-dotter-Jon övergång. Isotop-märkt föreningar har en nästan identisk övergripande fysikalisk-kemiska beteende jämfört med deras omärkt motsvarighet, är de idealiska interna standarder för MS/MS, vilket gör att en nästan matrix-oberoende analyten kvantifiering med en hög grad av noggrannhet15. Numera är många stabil isotop-märkt interna standarder som kan användas för småmolekylär kvantifiering, inklusive TDM av antimikrobiella medel, kommersiellt tillgängliga.
Kromatografisk separation av den antibiotiska analyter i protokollet beskrivs utförs med en analytisk C8 alkyl-kedja-längd bakåt-phase-kolonn (100 mm x 2,1 mm, 3 µm partikel-storlek). Under metodutvecklingen var de inre standard normaliserade matrix faktorerna för alla analyter mellan 94,6 och 105,4 procent, med en variationskoefficient för ≤8.3%14.

<table>
	<tbody>
		<tr>
			<td>cefepime hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>1097636</td>
			<td>USP Reference Standard</td>
		</tr>
		<tr>
			<td>meropenem trihydrate</td>
			<td>Sigma-Aldrich</td>
			<td>Y0001252</td>
			<td>EP Reference Standard</td>
		</tr>
		<tr>
			<td>ciprofloxacin</td>
			<td>Sigma-Aldrich</td>
			<td>17850</td>
			<td></td>
		</tr>
		<tr>
			<td>moxifloxacin hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>SML1581</td>
			<td></td>
		</tr>
		<tr>
			<td>linezolid</td>
			<td>Toronto Research Chemicals</td>
			<td>L466500</td>
			<td></td>
		</tr>
		<tr>
			<td>piperacillin sodium salt</td>
			<td>Sigma-Aldrich</td>
			<td>93129</td>
			<td></td>
		</tr>
		<tr>
			<td>cefepime-13C12D3 sulfate</td>
			<td>Alsachim</td>
			<td>C1297</td>
			<td>Isotope labelled internal standard for cefepime</td>
		</tr>
		<tr>
			<td>meropenem-D6</td>
			<td>Toronto Research Chemicals</td>
			<td>M225617</td>
			<td>Isotope labelled internal standard for meropenem</td>
		</tr>
		<tr>
			<td>ciprofloxacin-D8</td>
			<td>Toronto Research Chemicals</td>
			<td>C482501</td>
			<td>Isotope labelled internal standard for ciprofloxacin</td>
		</tr>
		<tr>
			<td>moxifloxacin-13C1D3 hydrochloride</td>
			<td>Toronto Research Chemicals</td>
			<td>M745003</td>
			<td>Isotope labelled internal standard for moxifloxacin</td>
		</tr>
		<tr>
			<td>linezolid-D3</td>
			<td>Toronto Research Chemicals</td>
			<td>L466502</td>
			<td>Isotope labelled internal standard for linezolid</td>
		</tr>
		<tr>
			<td>piperacillin-D5</td>
			<td>Toronto Research Chemicals</td>
			<td>P479952</td>
			<td>Isotope labelled internal standard for piperacillin</td>
		</tr>
		<tr>
			<td>methanol</td>
			<td>JT Baker</td>
			<td>8402</td>
			<td></td>
		</tr>
		<tr>
			<td>HPLC grade water</td>
			<td>JT Baker</td>
			<td>4218</td>
			<td></td>
		</tr>
		<tr>
			<td>formic acid</td>
			<td>Biosolve</td>
			<td>6914132</td>
			<td></td>
		</tr>
		<tr>
			<td>acetic acid</td>
			<td>Biosolve</td>
			<td>1070501</td>
			<td></td>
		</tr>
		<tr>
			<td>ammonium formate</td>
			<td>Sigma-Aldrich</td>
			<td>70221-25G-F</td>
			<td></td>
		</tr>
		<tr>
			<td>tert-Butyl methyl ether</td>
			<td>Merck</td>
			<td>101845</td>
			<td></td>
		</tr>
		<tr>
			<td>Fortis 3 &#956;m C8 100 * 2.1 mm</td>
			<td>Fortis</td>
			<td>F08-020503</td>
			<td></td>
		</tr>
		<tr>
			<td>Ti-PEEK-encased Prifilter (2 &#956;m)</td>
			<td>Chromsystems</td>
			<td>15011</td>
			<td></td>
		</tr>
		<tr>
			<td>2795 Alliance HPLC system</td>
			<td>Waters</td>
			<td>176000491</td>
			<td></td>
		</tr>
		<tr>
			<td>Quattro micro API Tandem Quadrupole System</td>
			<td>Waters</td>
			<td>720000338</td>
			<td></td>
		</tr>
		<tr>
			<td>QuanLynx 4.1 software</td>
			<td>Waters</td>
			<td>/</td>
			<td>Data evaluation software provided by the mass spectrometer manufacturer</td>
		</tr>
	</tbody>
</table>

multiplex therapeutic drug monitoring by isotope-dilution hplc-ms/ms of antibiotics in critical illnesses

Det finns en ständigt ökande efterfrågan på terapeutisk övervakning av antibiotika i många kliniska faciliteter, särskilt när det gäller genomförandet av sjukhuset antibiotika stewardship program.
I det nuvarande arbetet presenterar vi ett multiplex high-performance liquid chromatography-tandem masspektrometri (HPCL-MS/MS) protokoll för kvantifiering av cefepime, meropenem, ciprofloxacin, moxifloxacin, linezolid och piperacillin, vanligen används antibiotika inom intensivvården. Metoden validerades tidigare utförligt enligt riktlinje av Europeiska läkemedelsmyndigheten.
Efter en snabb prov rensning av analyter separeras i en C8 omvänd-phase HPLC-kolonn inom 4 minuter och kvantifieras med motsvarande stabil isotop-märkt interna standarder i elektrospray masspektrometri jonisering (ESI +) i flera reaktion tid övervakning (MRM). Den presenterade metoden använder en enkel instrumentering med enhetliga gaskromatografiska förhållanden, vilket möjliggör den dagliga och robust antibiotika terapeutisk övervakning i kliniska laboratorier. Kalibreringskurvan sträcker sig över den farmakokinetiska koncentrationsintervall, därmed inklusive antibiotika belopp nära den minsta hämmande koncentrationen (MIC) av mottagliga bakterier och maximal koncentration (Cmax) som erhålls med bolus administration behandlingsregimer. Utan att serumspädningen innan provet rensningen, kan arean under kurvan för en administrerad antibiotika erhållas genom flera mätningar.

Genom att använda protokollet beskrivs, avbildas ett typiskt kromatogram i figur 2. Enligt USA farmakopén (USP) kromatografi riktlinjer16, fastställdes den kolumn död volymen i det nuvarande systemet med ~0.22 mL och extra-kolumn volymen (inklusive injektor, slangar och kopplingar) med ~0.08 mL, ger en hold-up-volym ~0.30 ml. De beräkna kvarhållande faktorerna för alla analyter var 2,8 (för cefepime) - 4.2 (för piperacillin).<...

Watch this Scientific Journal Video about Multiplex Therapeutic Drug Monitoring by Isotope-dilution HPLC-MS/MS of Antibiotics in Critical Illnesses at JoVE.com

Multiplex Therapeutic Drug Monitoring by Isotope-dilution HPLC-MS/MS of Antibiotics in Critical Illnesses

Multiplex terapeutisk övervakning av Isotoputspädning HPLC-MS/MS av antibiotika i kritiska sjukdomar

Här presenterar vi ett tandem mass spectrometry-baserat protokoll för kvantifiering av vanliga antibiotika inom intensivvården, nämligen cefepime, meropenem, ciprofloxacin, moxifloxacin, linezolid och piperacillin.

There is an ever-increasing demand for the therapeutic drug monitoring of antibiotics in many clinical facilities, particularly with regard to the ...

Therapeutic drug monitoring of antibiotics is increasingly recognized as an important element for the implementation of national and local antibiotic stewardship programs. To meet this increasing demand of antibiotic therapeutic drug monitoring in medical facilities, we present a protocol for the quantification of antibiotics in circulation that aims to adjust the dosage to the susceptibility of the involved pathogens. The presented mass spectrometry-based protocol was optimized for the quantification of the most commonly used antibiotics in intensive care units, namely cefepime, meropenem, ciprofloxacin, moxifloxacin, linezolid, and piperacillin.Due to the broad calibration range, the method can also be used to calculate the pharmacokinetic area under the curve for an administered antibiotic. With a total turnaround time of approximately 30 minutes, the presented protocol is suitable for daily antibiotic therapeutic drug monitoring in clinical laboratories. First spike nine volumes of drug-free serum with one volume of previously prepared 10-fold concentrated spike solutions to obtain the serum calibrators zero through seven and quality controls A through D.For example, add 0.5 milliliters of spike solution to 4.5 milliliters of serum in a 10-milliliter polypropylene tube and incubate it for 15 minutes at four degrees Celsius on a roller mixer at 50 rpm.Use a repetitive pipette to generate 100-microliter aliquots of the calibrators and quality controls in 1.5-milliliter polypropylene tubes. Store the calibrators, quality controls, and previously prepared antibiotic stock solutions at minus 80 degrees Celsius for up to six months. After preparing six internal standard stock solutions, combine them in a 1.5-milliliter polypropylene tube to yield a five-fold concentrated internal standard mix.Add the desired internal standards to 965 microliters of 25%methanol in water. Store the internal standard stock solutions and the five-fold concentrated internal standard mix at minus 80 degrees Celsius. To prepare mobile phase A, add approximately 500 milliliters of HPLC grade water to a 1, 000-milliliter volumetric flask.Add one milliliter of formic acid, 10 milliliters of one-molar ammonium formate, and fill the flask to 1, 000 milliliters with HPLC grade water. Then transfer the mobile phase solution to a clean glass bottle. To prepare mobile phase B, add HPLC grade absolute methanol to a clean glass bottle.To prepare the purge solvent, add approximately 500 milliliters of distilled water to a 1, 000-milliliter volumetric flask. Add 70 milliliters of absolute methanol, one milliliter of formic acid, and fill the flask to 1, 000 milliliters with distilled water. Transfer the solvent to a clean glass bottle.Connect the bottles with mobile phases A and B and the purge solvent to the HPLC system. Make sure that the needle wash solvent tube is connected to the bottle containing mobile phase B.To prepare the precipitation agent, add 2.5 milliliters of methyl tert-butyl ether to a 25-milliliter volumetric flask and fill to 25 milliliters with absolute methanol. Place a C8 reversed phase column into the column chamber, then connect the column to the HPLC and mass spectrometer in the direction of the flow.In order to generate the sample list, open the corresponding sample measurement master file template and add the patient samples to be processed. Generate groups of up to 20 patient samples and flank them with the corresponding quality control pair. Using the Inlet file control software, set the Wet Prime function to 50%mobile phase A and 50%mobile phase B and wet prime for two minutes with a flow rate of one milliliter per minute.Next, refresh the syringe by executing six strokes of 600 microliters in the control software. To equilibrate the C8 reversed phase column, turn on the the flow in the inlet file and flush it with 7%mobile phase B and 93%mobile phase A for a minimum of five minutes, using a flow rate of 0.4 or 0.5 milliliters per minute. Visually verify the column temperature of 30 degrees Celsius.With a repetitive pipette, add 25 microliters of the internal standard mix to the thawed calibrator, QC sample, or patient serum in a 1.5-milliliter polypropylene tube and vortex the tube for a few seconds. Then incubate the mixture for five minutes at room temperature on a benchtop shaker. After incubation, add 150 microliters of the precipitation reagent to the sample internal standard mix with a repetitive pipette.Vortex the tube for a few seconds and then incubate for five minutes at room temperature on a benchtop shaker. Following this, centrifuge the suspension at 20, 000 times gravity in a tabletop centrifuge for 10 minutes at four degrees Celsius. Dilute the supernatant one in three with HPLC grade water in a glass vial with a micro insert.Then, place the sample in the HPLC autosampler. Manually start the HPLC-MS/MS analysis in the sample measurement control file. To process the samples, open the corresponding sample measurement control file, select the calibrators, quality controls, and patient samples and evaluate them with the antibiotics quantification method.Check whether the peaks for a specific analyte are properly integrated. Inspect the peaks for each calibrator, QC, and patient sample and manually reintegrate them at the baseline if necessary. Study the calibration curve and examine whether it fulfills linearity over the entire calibration range, a calibration coefficient greater than 0.99, a deviation of each calibration standard within plus or minus 15%of the nominal value except for the lower limit of quantification where a plus or minus 20%is required.Reject a calibration standard not complying with the above-mentioned criteria and reevaluate the calibration curve, including the regression analysis. Now, study the quality controls and examine whether the deviations are within plus or minus 15%of the nominal value. If the quality controls for a specific batch do not fulfill the quality criteria, the samples must be reinjected or even be reprocessed if significant deviations persist.Measured concentrations of a batch must also be evaluated whether they are plausible or not. If the concentration of a patient's sample exceeds the concentration of the highest calibrator, dilute the sample with distilled water up to one in five before the sample cleanup. Repeat the previous cleanup steps for that specific sample and reprocess it.The calculated retention factors for all analytes were 2.8 to 4.2. A sample chart list for the processed samples is shown here. The key parameter is the response gradually increasing with the analyte concentration due to the constant amount of added isotope-labeled internal standard.The calibration curve and quality controls for the example shown here fulfill all quality criteria. The coefficient of determination r-squared is greater than 0.995 and the deviation of the calibrators and the QC samples is within plus or minus 15%of the nominal value. The measured parent-to-daughter ion transitions show four peaks at the same retention time.The two upper peaks depict two transitions that are measured for the analyte of interest and the lower two peaks represent the transitions for the corresponding isotope-labeled internal standard. The first patient has a high serum trough level of 83.4 milligrams per liter of piperacillin that is also sufficient for problem pathogens. The second patient has a concentration of approximately 0.2 milligrams per liter, which is below the lowest calibrator.The third patient has low piperacillin trough concentration of only 5.3 milligrams per liter that is not sufficient for the majority of pathogens. The presented protocol is suitable for isotope dilution HPLC-tandem mass spectrometry-based quantification of cefepime, meropenem, ciprofloxacin, moxifloxacin, linezolid, and piperacillin in human serum. The method uses a rather simple instrument setup and will be there transferable to various mass spectrometry-based platforms.Chromatographic separation of the analytes of interest can also be obtained with a flow rate of 0.4 milliliters per minute as given in the present video. The described method can be used for pharmacokinetic studies as the calibration range allows both the quantification of concentrations close to the minimal inhibitory concentration of a susceptible pathogen as well as peak concentrations that are obtained with bolus administration regimens. After its development this method has been implemented in a mass spectrometry-based care facility for the antibiotic therapeutic drug monitoring in critically ill patients.Due to the fact that beta-lactam antibiotics are chemically unstable once they are dissolved, the quality of the pre-analytical phase such as sample storage plays a vital role in obtaining reliable quantification results. Don't forget that working with organic solvents is hazardous and precautions such as working in a fume should always be taken while preparing chromatography solvents and the precipitation reagent.

Research

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Medicine

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