Validation of a simple rapid high performance liquid chromatographic method for amniotic fluid lecithin/sphingomyelin ratios

D’Costa, Mario; Dassin, Roger; Bryan, Heather; Joutsi, P.

doi:10.1016/s0009-9120(85)80019-9

Cited by 8 publications

(10 citation statements)

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“…In the human infant the amount of tracheal fluid normally available in single aspirations is usually quite small and therefore a sensitive procedure is desirable. We have developed and clinically validated a sensitive and rapid HPLC method for LIS ratios in amniotic fluid (12) and now report on its application to the measurement of LIS ratios in tracheal fluid aspirates from newborn infants.…”

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confidence: 99%

“…A new analytical procedure for the determination of LIS in amniotic fluid has been described and is based on the separation oflecithin and sphingomyelin in Folch extracts of amniotic fluid by normal phase HPLe. This method has proved to be very sensitive, rapid and precise (12 Determining fetal pulmonary maturity by measuring LIS ratios in amniotic fluid as an index of surfactant activity has been a standard practice in obstetrics for many years (1). The presence of an immature LIS ratio in high risk pregnancies has influenced the timing of delivery in order to decrease the incidence of the RDS in the premature newborn infant.…”

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“…Chromatography was at ambient temperature with a flow rate of 1.9 ml/min and detection was by UV absorption at 205 nm. Lecithin and sphingomyelin eluted with retention times of 5 to 6 min and 8 to 9 min, respectively (12), and LIS ratios were computed with an online integrator (Hewlett-Packard 3390A). We used the same column manufacturer (Waters) in all our studies and observed no intercolumn variation.…”

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“…However, retention times and elution pattern may vary considerably with silica columns from other manufacturers. Details of the method precision (3-7% CV) and recovery of phospholipids (77-85%) have been described previously (12). For the analysis of LIS ratios in tracheal fluid the procedure has been modified as follows: tracheal fluid specimens were frozen at -20°C immediately upon receipt in the laboratory and stored for batch analyses.…”

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“…The diluted specimen was mixed to homogeneity by thoroughly vortexing for 5 min and then centrifuged at 1000 x g for 5 min to remove cells and debris. Clear supernatant (1.5 ml) was extracted with 1.5 ml of methanol and 3.0 ml of chloroform by the procedure described previously (12). Blood and meconium-stained specimens were rejected.…”

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Lecithin/Sphingomyelin Ratios in Tracheal Aspirates from Newborn Infants

1987

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MATERIALS AND METHODSmethod is noted for its laborious and time-consuming aspects and for the many variable factors which can influence the ratio (11). In the human infant the amount of tracheal fluid normally available in single aspirations is usually quite small and therefore a sensitive procedure is desirable. We have developed and clinically validated a sensitive and rapid HPLC method for LIS ratios in amniotic fluid (12) and now report on its application to the measurement of LIS ratios in tracheal fluid aspirates from newborn infants.Patients. Over an 18-method period tracheal fluid was aspirated from 115 newborn infants of high risk pregnancies during resuscitation at birth for the assessment of pulmonary maturity; GA ranged widely from 24 to 40 wk and birth weights from 570 to 4050 g. The numbers of babies related to GA groups and sex are listed in Table 1. Following resuscitation at delivery, chest radiographs determined the working clinical diagnosis for infants with clinical respiratory distress requiring intubation and assisted ventilation and for those needing only supplementary oxygen. RDS was diagnosed in 41 ventilated infants (GA 24 to 35 wk) based on x-ray presence of a diffuse reticular-granular pattern in the lung parenchyma and air bronchograms ( Table I); six of these infants died. Wet lung was noted in 19 infants (GA 24-34 wk) where chest radiographs showed normal lung aeration with increased perihilar markings and fluid in the fissures and 11 required mechanical ventilation (Table 1). Five infants were ventilated for other causes, three with focal pneumonias, one with pulmonary hypoplasia, and one with septicemia. Ten infants were ventilated for apnea of prematurity (GA <30 wk, birth weight <1420 g). These babies had normal initial chest xrays. A 32-wk infant who was ventilated for severe apnea died of herpes encephalitis at the age of 9 days (Table I).All samples were obtained within 3 h of delivery using no. 6 DeLee suction catheters (Argyle, St. Louis, MO) introduced either into the endotracheal tube in the intubated baby or directly below the cords into the trachea under direct vision laryngoscopy during resuscitation. In 27 of the intubated babies an additional 41 serial tracheal aspirates were obtained (43% within the 1st h oflife, 34% in the 2nd h, and 16% in the 3rd h) in order to assess changes in tracheal fluid LIS with time after birth . Amniotic fluid, sampled within 2 h ofdelivery, was comapred with tracheal fluid obtained in 24 infants. Twenty (83%) were amniocentesis samples obtained during Cesarian section deliveries while four were collected at the time of artificial rupture of the membranes within 2 h of delivery.LIS ratio determination . A new analytical procedure for the determination of LIS in amniotic fluid has been described and is based on the separation oflecithin and sphingomyelin in Folch extracts of amniotic fluid by normal phase HPLe. This method has proved to be very sensitive, rapid and precise (12). A 1O-lLm silica column (Waters microporasil 3.9 X 30 em) wa...

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Lecithin/Sphingomyelin Ratios in Tracheal Aspirates from Newborn Infants

1987

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Sphingolipidomics: Methods for the comprehensive analysis of sphingolipids

Haynes

Allegood

Park

et al. 2009

Journal of Chromatography B

118

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Sphingolipids comprise a highly diverse and complex class of molecules that serve as both structural components of cellular membranes and signaling molecules capable of eliciting apoptosis, differentiation, chemotaxis, and other responses in mammalian cells. Comprehensive or "sphingolipidomic" analyses (structure specific, quantitative analyses of all sphingolipids, or at least all members of a critical subset) are required in order to elucidate the role(s) of sphingolipids in a given biological context because so many of the sphingolipids in a biological system are interconverted structurally and metabolically. Despite the experimental challenges posed by the diversity of sphingolipid-regulated cellular responses, the detection and quantitation of multiple sphingolipids in a single sample has been made possible by combining classical analytical separation techniques such as high-performance liquid chromatography (HPLC) with state-of-the-art tandem mass spectrometry (MS/MS) techniques. As part of the Lipid MAPS consortium an internal standard cocktail was developed that comprises the signaling metabolites (i.e. sphingoid bases, sphingoid base-1-phosphates, ceramides, and ceramide-1-phosphates) as well as more complex species such as mono-and di-hexosylceramides and sphingomyelin. Additionally, the number of species that can be analyzed is growing rapidly with the addition of fatty acyl Co-As, sulfatides, and other complex sphingolipids as more internal standards are becoming available. The resulting LC-MS/MS analyses are one of the most analytically rigorous technologies that can provide the necessary sensitivity, structural specificity, and quantitative precision with high-throughput for "sphingolipidomic" analyses in small sample quantities. This review summarizes historical and state-of-the-art analytical techniques used for the for the identification, structure determination, and quantitation of sphingolipids from free sphingoid bases through more complex sphingolipids such as sphingomyelins, lactosylceramides, and sulfatides including those intermediates currently considered sphingolipid "second messengers". Also discussed are some emerging techniques and other issues remaining to be resolved for the analysis of the full sphingolipidome.

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