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Editor-in-chief
Maria Stella Graziani
Deputy Director
Martina Zaninotto
Associate Editors
Ferruccio Ceriotti
Davide Giavarina
Bruna Lo Sasso
Giampaolo Merlini
Martina Montagnana
Andrea Mosca
Paola Pezzati
Rossella Tomaiuolo
Matteo Vidali
EIC Assistant
Francesco Busardò
International Advisory Board
Khosrow Adeli Canada
Sergio Bernardini Italy
Marcello Ciaccio Italy
Eleftherios Diamandis Canada
Philippe Gillery France
Kjell Grankvist Sweden
Hans Jacobs The Netherlands
Eric Kilpatrick UK
Magdalena Krintus Poland
Giuseppe Lippi Italy
Mario Plebani Italy
Sverre Sandberg Norway
Ana-Maria Simundic Croatia
Tommaso Trenti Italy
Cas Weykamp The Netherlands
Maria Willrich USA
Paul Yip Canada
Publisher
Biomedia srl
Via L. Temolo 4, 20126 Milano
Responsible Editor
Giuseppe Agosta
Editorial Secretary
Chiara Riva
Biomedia srl
Via L. Temolo 4, 20126 Milano
Tel. 0245498282
email: biochimica.clinica@sibioc.it
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ISSN print: 0393 – 0564
ISSN digital: 0392- 7091
BC: Articoli scritti da S. Persichilli
Validazione dei metodi quantitativi bioanalitici in spettrometria di massa: regole e protocolli sperimentali
Bioanalytical method validation of quantitative mass spectrometry based assay: experimental protocols and regulations
<p>Liquid chromatography-mass spectrometry (LC-MS) technique has rapidly extended the spectrum of clinical chemistry methods during the past decade and it has became a standard tool for endogenous and exogenous substances in research laboratories as well as in many clinical laboratories. To date, many guidelines for bioanalytical method validation have been published, but there is still the need to have specific guidelines and experimental protocols. In particular, we need to further focus and investigate the overall clinical aspects of spectrometry method validation, for both exogenous and endogenous molecules. The aim of the present document is to suggest specific experimental regulations and protocols for bioanalytical LC-MS method validation for exogenous and endogenous molecules, inspired by actual international guidelines and integrated with practical tips to obtain better performance on methods used in routine clinical practice. Here we have introduced new concepts such as “normalized matrix effect”, and protocols for method validation using “pathological samples” and “altered samples”, as these are the most commonly samples encountered in laboratory routine. In conclusion, the new rules and protocols reported in this work do not intend to replace international guidelines; we believe that these rules have to be considered besides the available guidelines with the aim to help the LC-MS users by recommending a number of experimental steps to be performed during each method/kit validation.</p>
<p>A 22-year-old male with recurrent bilateral stone disease and positive family history for stones was referred in March 2015 to the Metabolic Stone Clinic of the Policlinico Gemelli Hospital; a metabolic assessment revealed a number of abnormalities including hypercalciuria, hypocitraturia and abnormally high fasting urine pH. The previously expelled urinary calculus was sent to the clinical laboratory for the biochemical analysis by the infrared method. Fourier transform infrared spectroscopy (FT-IR) analysis identifying brushite and carbapatite, together with the finding of high urine pH, raised the suspicion of distal renal tubular acidosis which was subsequently confirmed by the urinary acidification test. The semiquantitative method of stone composition analysis would have yielded a mixture of calcium, ammonium, phosphate and magnesium, thus likely directing the diagnostic work-up, together with the abnormally high urine pH, towards infectious stones (e.g., by urease-producing bacteria). The information obtained by FT-IR analysis allowed clinicians to correctly hypothesize a urinary acidification deficit, which was subsequently treated with potassium citrate supplements.</p>
Biochimica Clinica ; 41(2) e13-e14 Casi clinici - Case report
Sviluppo di un metodo in cromatografia liquida associata alla spettrometria di massa tandem per valutare la permeabilità intestinale nella pratica clinica
Development of a liquid chromatography-tandem mass spectometry (LC-MS/MS) method to evaluate intestinal permeability in clinical practice
<p>The intestinal permeability test is a non-invasive method for assessing gastrointestinal mucosal integrity. The lactulose/mannitol ratio is used to assess the intestinal barrier function. Aim of this work was to develop a rapid and robust method for the simultaneous analysis of lactulose and mannitol in urine by LC-MS/MS. Calibration curves were prepared in water:acetonitrile 20:80. Raffinose was used as internal standard at 25 mg/L in water:acetonitrile 20:80. 50 μL of urine sample was added to 450 μL of internal standard solution, and then vortexed and centrifuged. A 200 μL aliquot was transferred into a vial and placed in the autosampler for LCMS/MS analysis. The chromatographic separation was performed using a Phenomenex Luna NH2 column operating at a flow rate of 200 μL/min and eluted with a linear gradient from 20 to 80 water in acetonitrile. The total run time was 16 min. The MS operates in electrospray negative mode. The method was validated according to European Medicine Agency guidelines. Linearity ranged from 10 to 800 mg/L for mannitol and from 2.5 to 400 mg/L for lactulose, respectively. Inter-assay imprecision (expressed as CV) was <13% for both analytes. Recovery was >85%.</p>
<p>Nephrolithiasis is a common disease occurring in both industrialized and developing countries and mainly affecting adults aged 20-60 years. The determination of crystalline and molecular composition and the quantification of all stone components are helpful to establish the etiology of urinary stone disease. Different methodologies exist for the analysis of renal stones. In our laboratory, we used the chemical spot test, which is the most popular method for routine stone analysis. However, this method is relatively inaccurate and does not allow distinguishing between the crystalline phases. In this paper, we evaluated the quantitative FT-IR spectroscopy technique for a possible introduction in our laboratory. We analyzed 100 urinary stones, divided into 3 groups: pure stones, mixed stones, and pure stones with substances in trace. Results of each group were compared with those obtained with chemical spot test. A total or partial disagreement between the two methods was observed in 59% of pure stones, in 86.6% of mixed stones and in 100% of pure stones with substances in trace, respectively, suggesting the replacement of the chemical test with FT-IR spectroscopy.</p>
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