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Maria Stella Graziani

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Martina Zaninotto

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Ferruccio Ceriotti
Davide Giavarina
Bruna Lo Sasso
Giampaolo Merlini
Martina Montagnana
Andrea Mosca
Paola Pezzati
Rossella Tomaiuolo
Matteo Vidali

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Sergio Bernardini Italy
Marcello Ciaccio Italy
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Giuseppe Lippi Italy
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Ana-Maria Simundic Croatia
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Tommaso Trenti Italy
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Paul Yip Canada


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Giuseppe Agosta

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Arianna Lucini Paioni
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email: biochimica.clinica@sibioc.it



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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 &ldquo;normalized matrix effect&rdquo;, and protocols for method validation using &ldquo;pathological samples&rdquo; and &ldquo;altered samples&rdquo;, 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>
Biochimica Clinica ; 42(1) 51-61
Documenti - Documents
 
Utilità della spettroscopia all’infrarosso per l’analisi dei calcoli urinari
Usefulness of infrared spectroscopy for stone analysis: a case study
<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 &mu;L of urine sample was added to 450 &mu;L of internal standard solution, and then vortexed and centrifuged. A 200 &mu;L 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 &mu;L/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 &lt;13% for both analytes. Recovery was &gt;85%.</p>
Biochimica Clinica ; 41(1) 060-063
Contributi scientifici - Scientific papers
 
Ruolo della spettroscopia infrarossa in trasformata di Fourier nella valutazione della calcolosi urinaria
Role of Fourier transform infrared (FT-IR) spectroscopy in the evaluation of urinary stone disease
<p>Nephrolithiasis is a common disease occurring in both industrialized and developing countries and mainly affecting&nbsp;adults aged 20-60 years. The determination of crystalline and molecular composition and the quantification of all&nbsp;stone components are helpful to establish the etiology of urinary stone disease. Different methodologies exist for the&nbsp;analysis of renal stones. In our laboratory, we used the chemical spot test, which is the most popular method for&nbsp;routine stone analysis. However, this method is relatively inaccurate and does not allow distinguishing between the&nbsp;crystalline phases. In this paper, we evaluated the quantitative FT-IR spectroscopy technique for a possible&nbsp;introduction in our laboratory. We analyzed 100 urinary stones, divided into 3 groups: pure stones, mixed stones, and&nbsp;pure stones with substances in trace. Results of each group were compared with those obtained with chemical spot&nbsp;test. A total or partial disagreement between the two methods was observed in 59% of pure stones, in 86.6% of mixed&nbsp;stones and in 100% of pure stones with substances in trace, respectively, suggesting the replacement of the chemical&nbsp;test with FT-IR spectroscopy.</p>
Biochimica Clinica ; 40(1) 28-32
Contributi scientifici - Scientific Papers