ABSTRACT
Ion-exchange chromatography with ninhydrin detection remains the gold standard for detecting inborn errors of amino acid catabolism and transport. Disadvantages of such analysis include long chromatography times and interference from other ninhydrin-positive compounds. The aim of this project was to develop a more rapid and specific technique using liquid chromatography/tandem mass spectrometry (LC/MS/MS). Optimal fragmentation patterns for 32 amino acids were determined on a triple quadrupole mass spectrometer following butylation. Chromatographic characteristics of each of the amino acids were determined using C8 reversed-phase chromatography with 20% acetonitrile/0.1% formic acid as isocratic mobile phase. Quantitation using eleven deuterated internal standards was compared to cation exchange and ninhydrin detection on a Beckman 7300 system. Following methanol extraction and butylation, determination of 32 amino acids required 20 min. The dynamic range of each amino acid was generally 1-1000 micromol/L. Imprecision ranged from 7 to 23% (CV) over 6 months and recovery ranged from 88-125%. Deming regression with the Beckman 7300 yielded slopes from 0.4-1.2, intercepts from -21 to 65 micromol/L, correlation coefficients from 0.84-0.99 and Syx from 2-125 micromol/L. Isobaric amino acids were separated by chromatography (e.g. leucine, isoleucine) or by unique fragmentation (e.g., alanine, beta-alanine). LC/MS/MS is comparable to traditional LC-ninhydrin detection. Mass spectral detection shortens analysis times and reduces potential for interference in detecting inborn metabolic errors.
Subject(s)
Amino Acids/chemistry , Chromatography, Liquid/methods , Ninhydrin/chemistry , Tandem Mass Spectrometry/methods , Amino Acids/blood , Amino Acids/urine , Chromatography, Ion Exchange/methods , HumansABSTRACT
BACKGROUND: A new total bilirubin (B(T)) method, based on multiple wavelength absorbance measurements, and an algorithm to calculate concentration, were evaluated for accuracy in specimens containing variable amounts of unconjugated bilirubin (B(U)), conjugated bilirubin (B(C)) and delta (protein-bound) bilirubin (B(D)). METHODS: Quantitation of B(U), B(C), and B(T) (with calculation of B(D)) using a Vitros 250 analyzer served as the comparison method. RESULTS: Analysis of neonatal specimens using a preliminary algorithm yielded good overall agreement with the Vitros B(T) method, but there was considerable variation in the agreement for individual specimens. When specimens from adults selected to yield a range of B(C) and B(D) levels were analyzed, the preliminary algorithm underestimated B(T). Refinement of the method in the form of a finalized algorithm resulted in elimination of the negative bias seen with specimens with high B(D) and B(C) levels, and better agreement for individual neonatal specimens. CONCLUSIONS: This new method overcomes the limitations observed in earlier spectrophotometric methods, and provides accurate results in specimens containing a range of bilirubin forms.
Subject(s)
Bilirubin/blood , Blood Gas Analysis/methods , Spectrophotometry, Atomic/methods , Adult , Algorithms , Bilirubin/chemistry , Humans , Infant, Newborn , Linear Models , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
Analisadores compactos, apropriados para testes em pacientes à mão, avaliam o hematócrito pela medida da condutividade do sangue não diluído. Nós avaliamos a exatidão do resultado de hematócrito de um determinado analisador (Instrumentation Laboratory BGE Analyzer) em comparação com um automático contador eletrônico de partículas (CEP) e volume de células sedimentadas (VCS) microhematócrito. Quando amostras (n = 34) de pacientes externos e de enfermaria foram analisadas por todos os três métodos, o analisador AEG estava de acordo com os dois hematócritos CEP e VCS (AEG = 1,00 VCS + 0,3%, Sy/x = 1,6% ; AEG = 1,04 CEP + 0,4%, Sy/x = 2,0%), indicando que todos os três métodos têm performances similares na maioria dos pacientes. Entretanto, um paciente com osmolalidade plasmática aumentada mostrou significante decréscimo nos hematócritos AEG e VCS em comparação ao método CEP. As diferenças nas medidas do hematócrito podiam ser reproduzidas pela adição de solutos ao sangue in vitro ou pela modificação de osmolalidade plasmática de ratos "in vivo". Amostras de pacientes submetidos a uma cirurgia cardíaca, cujo sangue tinha grandes mudanças na concentração de proteína, mostrou discrepância entre hematócritos pela condutividade e outros métodos; efeitos similares poderiam ser produzidos pelas mudanças na concentração de proteína ou pela adição "in vitro" de polietileno glicol. Nós concluímos que medidas de condutividade fornecem resultados exatos do hematócrito para sujeitos normais fisiologicamente, mas não para alguns pacientes sob cuidado intensivo e pacientes cirúrgicos.
