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

Yazarlar Dr Schütze sunulan Yöntem kurma onun yardım için teşekkür ederiz ve Dr Zoller değerli giriş için uygun kalibrasyon aralığı ile ilgili. Yazarlar da kütle spektrometresi tesisinin teknik personel kabul.

Not: Bu bir duman mahallede metanol gibi organik çözücü işlerken çalışmak için tavsiye edilir. Tüm arabellekleri ve mobil aşamaları hacimsel şişeler içinde hazır olun. Başka şekilde belirtildiği takdirde çözümleri oda sıcaklığında 1 ay sonra hazırlık için saklanır.
1. hazırlanması Kalibratörler ve kalite kontrol örnekleri
Not: Stok ve spike çözümleri hazırlanması için karşılık gelen bir veri analizi sayfasında Ek dos...

<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>

Bu makale, biz ICU19, cefepime, meropenem, Siprofloksasin, moxifloxacin, linezolid, sık kullanılan antibiyotikler miktar için basit ve sağlam tandem kütle spektrometresi tabanlı yöntemi için protokol raporu ve piperacillin14. Bir elektronik tablo el yazması antibiyotik hisse senedi çözümleri, kalibratörler ve kalite kontrol, antibiyotikler saflığı ve onların counterions molekül ağırlığı dikkate alarak hazırlanması için eşlik eder. Antibiyotiklerin...

Her ne kadar antibiyotik tıp pratiği devrim, şiddetli bakteriyel enfeksiyonlar önde gelen nedenidir morbidite ve mortalite kritik hastalıklar1kalır. Bu bağlamda, komut istemi bir uygun anti infective yeterli dozda en üstte yer alan önemli hastalık denetim2için bir ilçedir.
Kanıt büyüyen bir vücudun geniş spektrumlu antibiyotik ile ampirik tedavi hasta nüfus karmaşıklığı ile giderek daha sorunlu hale geliyor gösterir. Bu özellikle yoğun bakım üniteleri (ICU), nerede anahtar farmakokinetik (PK) parametreleri muazzam bir arası bireysel değişkenliği sık3,4görülmektedir doğrudur. Buna göre ICU hastaların yakın alt terapötik düzeylerinin yetersiz tedavi başarı5,6tehlikesi ile etkilenir. Sonra tekrar, hastalara gereksiz yere hiçbir klinik yarar7ile ciddi advers olaylar neden olabilir aşırı yüksek antibiyotik konsantrasyonları maruz kalır. Antibiyotik kötüye ve yetersiz dozaj halk sağlığı8için büyüyen bir tehdit haline geliyor antibiyotik direnci yayılması da yol açtı.
Mümkün olduğunca uzun süre antibiyotik kullanımı artırmak için ve kendi effectivenessas korumak için Dünya Sağlık Örgütü 20159antimikrobiyal direnç bir küresel eylem planı başlattı. Antibiyotik yönetim programları Ulusal Halk Sağlığı stratejileri10hasta bakımı11 ve aynı zamanda kalitesini önemli ölçüde artırmak için klinisyenler yardım, ihtiyatlı antibiyotik kullanımının temel bir temel taşı teşkil antibiyotik direnci12azaltılması. Antimikrobiyal tedavi edici ilaç uygulanması yoluyla bireysel hastalarda dozlama (TDM) izleme bir anahtar bu bağlam13' te aracıdır.
Bugüne kadar piyasada bulunan TDM deneyleri glycopeptide antibiyotik ve aminoglikozitler için kullanılabilirdir. Diğer sınıflardan maddelerin miktar yaygın bir in-house Yöntem geliştirme veya hantal olabilir doğrulama gerektirir. Biz, bu nedenle, kendi klinik ilgili toplama aralıkları14içinde yoğun bakımda antibiyotikler en uygun miktar için kullanılan güçlü bir kütle spektrometresi tabanlı tahlil için protokol ayrıntılı olarak mevcut. Yöntem kütle spektrometresi tesisimizde son zamanlarda kuruldu ve o zamandan beri rutin yoğun bakımda TDM için uygulanan. Yordamı kütle spektrometresi yetenekleri ile birçok tesislerinde antibiyotik TDM hızlı uygulama için izin veren bir üniforma örnek Temizleme ile kolay ve basittir analitik ayarını kullanır.
Burada açıklanan protokol cefepime, meropenem, Siprofloksasin, moxifloxacin, linezolid ve piperacillin bir tandem ile birlikte İzotop seyreltme sıvı kromatografi (LC) kullanarak insan serumda miktar için optimize edildi kitle spektrometresi (MS/MS). İzotop seyreltme için LC-MS/MS metodoloji kararlı izotop etiketli bileşikler (Örneğin, serum) belirli bir matris ile ilgi bir örnek eklenir. İzotop etiketli standartları yani analit doğal molekül ve bir üst iyon kızı iyon geçiş olarak adlandırdığı parçalanma ürünlerinin farklı molekül ağırlıkları nedeniyle ilgi onların etiketlenmemiş karşılığından ayrılır. İzotop etiketli bileşikler için etiketlenmemiş kendi meslektaşı karşılaştırıldığında hemen hemen aynı bir genel olarak fizikokimyasal davranış var onlar MS/MS, hemen hemen matris bağımsız analit miktar yüksek düzeyde izin için ideal kendi standartlarına şunlardır doğruluk15. Günümüzde, antimicrobials, TDM dahil olmak üzere küçük molekül miktar için kullanılabilecek pek çok kararlı izotop etiketli kendi standartlarına ticari olarak kullanılabilir.
Açıklanan protokolündeki antibiyotik analitler kromatografik ayırma bir analitik C8 alkil-zincir-uzunluk ters fazlı sütun (100 mm x 2.1 mm, 3 µm parçacık boyutu) gerçekleştirilir. Yöntem geliştirme sırasında bütün analitler için iç standart normalleştirilmiş matris etkenler %94.6 ve %105.4, ≤8.3%14varyasyon katsayısı ile arasında yapıldı.

<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

Tedavi edici ilaç antibiyotik hastane antibiyotik yönetim programlarının uygulanması açısından özellikle birçok klinik tesislerinde izlemek için artan bir talep var.
Geçerli çalışma, biz cefepime, meropenem, Siprofloksasin, moxifloxacin, linezolid ve piperacillin, yaygın olarak kullanılan miktar için bir çok katmanlı yüksek performanslı sıvı kromatografi-tandem kütle spektrometresi (HPCL-MS/MS) iletişim kuralı mevcut yoğun bakım ünitelerinde antibiyotik. Yöntemi daha önce kapsamlı bir şekilde Avrupa İlaç Ajansı kılavuz göre doğrulandı.
Hızlı örnek temizleme sonra analitler 4 dakika içinde bir C8 ters fazlı HPLC sütun ayrılmış ve karşılık gelen kararlı izotop etiketli iç standartları ile electrospray iyonlaşma (ESI +) kütle spektrometresi birden çok tepki sayılabilir (MRM) izlemeye zaman. Sunulan yöntemi ile Tekdüzen kromatografik koşulları ayarlama, günlük ve güçlü antibiyotik tedavi edici ilaç klinik laboratuarlarda izlemek için izin veren bir basit araçları kullanır. Kalibrasyon eğrisi böylece minimum inhibitör konsantrasyonu (MIC) yakın antibiyotik miktarda duyarlı bakteri ve bolus ile elde edilen en yüksek konsantrasyonları (Cmax) dahil olmak üzere farmakokinetik konsantrasyon aralığı aşıyor Yönetim rejimleri. Örnek temizleme önce serum seyreltme zorunluluk yönetilen bir antibiyotik için eğri altındaki alan birden fazla ölçümleri elde edilebilir.

Açıklanan iletişim kuralını kullanarak, tipik bir kromatografik Şekil 2' de tasvir edilir. Amerika Birleşik Devletleri Pharmacopeia (USP) Kromatografi yönergeleri16göre mevcut sistemde sütun ölü cilt ~0.22 mL ve (enjektör, boru ve bağlayıcıları içeren) ekstra sütunlu birimle ~0.08 mL, kararlıydı veren ~0.30 mL soygun hacmi. Bütün analitler için hesaplanan saklama faktörler (için cefepime) - 2,8 vardı 4.2 (iç...

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

İzotop seyreltme HPLC-MS/MS kritik hastalıklar içinde antibiyotik ile tedavi edici ilaç izleme multiplex

Burada bir tandem kütle spektrometresi tabanlı protokol sık kullanılan antibiyotikler miktar yoğun bakım üniteleri, yani cefepime, meropenem, Siprofloksasin, moxifloxacin, linezolid ve piperacillin mevcut.

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

JoVE Journal

Medicine

Bronchial Thermoplasty:  A Novel Therapeutic Approach to Severe Asthma

Bronş Thermoplasty: Şiddetli Astım Yeni Bir Tedavi Yaklaşımı

Bronchial thermoplasty is a non-drug procedure for severe persistent asthma that delivers thermal energy to the airway wall in a precisely controlled ...

Tıp

An Affordable HIV-1 Drug Resistance Monitoring Method for Resource Limited Settings

Kaynak Sınırlı Ayarlar için uygun bir HIV-1 İlaç Direnci İzleme Yöntemi

HIV-1 drug resistance has the potential to seriously compromise the effectiveness and impact of antiretroviral therapy (ART). As ART programs in ...

A Simple Critical-sized Femoral Defect Model in Mice

Farelerde Basit Kritik ölçekli Femoral Hata Modeli

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all ...

Therapeutic Effectiveness of a Dietary Supplement for Management of Halitosis in Dogs

Köpeklerde Ağız kokusunun Yönetimi için Diyet Supplement Terapötik Etkinliği

Halitosis is a common complaint involving social and communicational problems in humans and also affects the pet-owner relationship. In this randomized ...

Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring

Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring

Için Polikistik Böbrek Hastalığında Küçük Kemirgen modelleri Ultra yüksek Saha MRI Kullanımı<em&gt; In Vivo</em&gt; Fenotiplendirme ve İlaç İzleme

Several in vivo pre-clinical studies in Polycystic Kidney Disease (PKD) utilize orthologous rodent models to identify and study the genetic and molecular ...

Establishment of a Segmental Femoral Critical-size Defect Model in Mice Stabilized by Plate Osteosynthesis

Farelerde bir Segmental Femoral Kritik boyutlu Defekt Modelinde kurulması Plaka Osteosentez tarafından Stabilize

The use of tissue-engineered bone constructs is an appealing strategy to overcome drawbacks of autografts for the treatment of massive bone defects. As a ...

Standardized Hemorrhagic Shock Induction Guided by Cerebral Oximetry and Extended Hemodynamic Monitoring in Pigs

Domuzlarda serebral oksimetre ve genişletilmiş hemodinamik Izleme güdümlü standartlaştırılmış hemorajik şok Indüksiyon

Hemorrhagic shock ranks among the main reasons for severe injury-related death. The loss of circulatory volume and oxygen carriers can lead to an ...

Assessing Therapeutic Angiogenesis in a Murine Model of Hindlimb Ischemia

Hindekstremite Iskemi bir Murine modelinde terapötik anjiyogenezi değerlendirilmesi

Critical limb ischemia (CLI) is a serious condition that entails a high risk of lower limb amputation. Despite revascularization being the gold-standard ...

A Rat Carotid Artery Pressure-Controlled Segmental Balloon Injury with Periadventitial Therapeutic Application

Periadventitial Terapötik Uygulama ile Sıçan Karotis Arter Basınç Kontrollü Segmental Balon Yaralanması

Cardiovascular disease remains the leading cause of death and disability worldwide, in part due to atherosclerosis. Atherosclerotic plaque narrows the ...

Validated LC-MS/MS Panel for Quantifying 11 Drug-Resistant TB Medications in Small Hair Samples

Küçük Saç Örneklerinde 11 İlaca Dirençli TB İlaçların Ölçülmesi için Doğrulanmış LC-MS/MS Paneli

Drug resistant-tuberculosis (DR-TB) is a growing public health threat, and assessment of therapeutic drug levels may have important clinical benefits. ...