Journal of Chromatography B, 993 (2015) 81–85
Contents lists available at ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Prediction of fetal lung maturity using the lecithin/sphingomyelin (L/S) ratio analysis with a simplified sample preparation, using a commercial microtip-column combined with mass spectrometric analysis Ho-Seok Kwak a , Hee-Jung Chung a,∗ , Young Sik Choi b , Won-Ki Min c , So Young Jung d a Department of Laboratory Medicine, Cheil General Hospital & Women’s Healthcare Center, Dankook University College of Medicine, Seoul, Republic of Korea b Department of Chemistry, Inha University, Incheon, Republic of Korea c Department of Laboratory Medicine, University of Ulsan College of Medicine & Asan Medical Center, Seoul, Republic of Korea d Department of Surgery, Hallym University Kangnam Sacred Heart Hospital, Seoul, Republic of Korea
a r t i c l e
i n f o
Article history: Received 12 December 2014 Received in revised form 20 April 2015 Accepted 10 May 2015 Available online 14 May 2015 Keywords: Lecithin/sphingomyelin ratio Fetal lung maturity Cation-exchange resin ESI-MS/MS MALDI-TOF
a b s t r a c t Fetal lung maturity is estimated using the lecithin/sphingomyelin ratio (L/S ratio) in amniotic fluid and it is commonly measured with thin-layer chromatography (TLC). The TLC method is time consuming and technically difficult; however, it is widely used because there is no alternative. We evaluated a novel method for measuring the L/S ratio, which involves a tip-column with a cation-exchange resin and mass spectrometry. Phospholipids in the amniotic fluid were extracted using methanol and chloroform. Choline-containing phospholipids such as lecithin and sphingomyelin were purified by passing them through the tip-column. LC–MS/MS and MALDI-TOF were used to directly analyze the purified samples. The L/S ratio by mass spectrometry was calculated from the sum peak intensity of the six lecithin, and that of sphingomyelin 34:1. In 20 samples, the L/S ratio determined with TLC was significantly correlated with that obtained by LC–MS/MS and MALDI-TOF. There was a 100% concordance between the L/S ratio by TLC and that by LC–MS/MS (kappa value = 1.0). The concordance between the L/S ratio by TLC and that by MALDI-TOF was also 100% (kappa value = 1.0). Our method provides a faster, simpler, and more reliable assessment of fetal lung maturity. The L/S ratio measured by LC–MS/MS and MALDI-TOF offers a compelling alternative method to traditional TLC. © 2015 Published by Elsevier B.V.
1. Introduction Respiratory distress syndrome (RDS) is a major contributor to neonatal morbidity and mortality worldwide, affecting approximately 1% of all live births and 10% of all preterm infants [1]. RDS is the most common respiratory disorder of premature newborns. It is diagnosed in ∼70% of very low birth weight infants (501–1500 g) in the USA [2]. The preterm birth rate has risen in most developed
∗ Corresponding author at: Department of Laboratory Medicine, Cheil General Hospital & Women’s Healthcare, Center and Dankook University College of Medicine, 1-19 Mukjeong-dong, Jung-gu, Seoul 100-380, Republic of Korea. Tel.: +82 2 2000 7290; fax: +82 2 2000 7780. E-mail address: [email protected] (H.-J. Chung). http://dx.doi.org/10.1016/j.jchromb.2015.05.012 1570-0232/© 2015 Published by Elsevier B.V.
countries [2]. Despite the decreased use of the fetal lung maturity (FLM) test, it is still very important for planning deliveries in the preterm interval [3,4]. Through 2011, the most widely used FLM test was the TDx Fetal Lung Maturity II (Abbott Diagnostics, Il, USA). The inability to order the TDx Fetal Lung Maturity II test will likely increase the number of requests for the lecithin/sphingomyelin (L/S) ratio and the lamellar body count (LBC) [3]. The L/S ratio was the first established technique to estimate adequate fetal surfactant production [5]. Currently, the L/S ratio of amniotic fluid is most often measured using thin-layer chromatography (TLC). The TLC method is a one-dimensional procedure often performed using the Fetal-Tek 200 kit from Helena Laboratories [6]. However, it is both technically difficult and time-consuming.
82
H.-S. Kwak et al. / J. Chromatogr. B 993 (2015) 81–85
Previously, we proposed new methods to measure the L/S ratio including liquid chromatography tandem mass spectrometry (LC–MS/MS) and matrix-assisted laser desorption and ionization time of flight mass spectrometry (MALDI-TOF MS) [7]. The aim of this study was to assess the ability of these new methods to predict FLM and to compare their performance to that of conventional TLC. 2. Material and methods 2.1. Materials and reagents The Micro tip-column (packing material: strong cation) was purchased from Harvard Apparatus (Holliston, MA, USA) and 2,5dihydroxybenzoic acid (DHB) was obtained from Sigma–Aldrich (Louis, MO, USA). All other chemicals of analytical grade, or HPLC grade, were obtained from J.T. Baker (Phillipsburg, NJ, USA). The Fetal-Tek 200 kit was purchased from Helena Laboratories (Beaumont, TX, USA). 2.2. Sample collection The consecutive series of physician-ordered L/S ratio samples, collected during a one year period, were examined in Asan Medical Center (Seoul, Republic of Korea). FLM testing was ordered for women presenting with symptoms of preterm labor and for asymptomatic women presenting before scheduled cesarean sections. Amniotic fluid obtained by amniocentesis was collected in EDTA tubes and plain tubes from non-diabetic patients with gestational ages between 21 and 35 weeks. RDS symptoms were confirmed by review of the newborn records. After routine analysis, the samples were stored at −20 ◦ C until they were analyzed by mass spectrometry (MS). 2.3. Measurement of L/S ratio by TLC We routinely performed the Fetal-Tek 200 procedure according to the manufacturer’s instructions, without any modifications. The amniotic fluid in the plain tube was extracted with a mixture of chloroform and methanol and applied across the channel of a TLC plate. The mobile phase is a mixture of chloroform, methanol, 2-propanol, triethylamine, and water. The lecithin and sphingomyelin bands were well separated from the other phospholipids and neutral lipids. In order to be visualized, plates were prepared with phosphoric acid containing a cupric acetate catalyst. The lecithin-to-sphingomyelin ratio was calculated by scanning the lecithin and sphingomyelin bands on the TLC plates.
with a sulphopropyl group. In order to equilibrate and rinse the material in the tip, 250 uL of 90% acetonitrile (ACN) with 0.1% formic acid was added to the top of the tip and then was dispensed the fluid by syringe. Next, 250 uL of extracted solution (lower phase solution) was added to the top of the tip and then slowly dispensed the fluid. To elute the phospholipids, 150 uL 90% ACN with 0.1% formic acid was added to the top of the tip and then slowly dispensed the fluid. In the LC–MS/MS analysis, samples were quantified by direct flow injection. The flow solvent (90% ACN with 0.1% formic acid) was added at a constant flow of 50 uL/min by the PerkinElmer series 200. The triple quadrupole mass spectrometry (API 2000, MDX/Sciex, Canada) was operated using the following settings: capillary voltage, +5.5 kV; temperature in ion source, 300 ◦ C; collision energy, 40; collision gas, nitrogen at a pressure of 1.5 10–5 Torr. A precursor ion scan of m/z 184.1 specific for phosphocholinecontaining lipids was used for quantification [9]. Data analysis was performed using the Analyst software. In the MALDI-TOF MS analysis, matrix stock solutions were made up in 80% methanol at a concentration of 10 mg/mL. A 1 uL aliquot of each sample solution was mixed with 10 uL of a matrix solution. A 1 uL aliquot of the final solution of sample and matrix was manually deposited onto a stainless steel plate. The sample plate was rapidly dried under a warm air stream to improve the homogeneity of crystallization. The MALDI-TOF mass spectra were acquired on a Voyager DE-STR workstation (PerSeptive Biosystems, Framingham, MA). This system utilizes a pulsed nitrogen laser, emitting 337 nm at 3 Hz. The MALDI-TOF was operated using the following settings: accelerating voltage, +20 kV; grid voltage, 60%; extraction delay time, 100 n sec; low mass gate, 400 Da. The “low mass gate” was turned on to prevent the detector from becoming saturated by ions from the matrix. For each mass spectrum, 150 single laser shots were averaged. In order to enhance the spectral resolution, all spectra were recorded in the reflector mode. Data analysis was performed using Data Explorer TM. 2.6. Statistical analysis Data were analyzed using the SPSS 18 statistical software (SPSS Inc., Chicago, IL, USA). The relationship among the various parameters was tested and expressed using Pearson’s correlation coefficient. P-values < 0.05 were considered statistically significant. Cohen’s Kappa was used to assess agreement between each method’s measure of fetal lung maturity (agreement: 0.6, substantial to almost perfect) [10].
2.4. Measurement of the lamellar body count (LBC)
3. Results and discussion
The Sysmex XE-2100 (Sysmex, Kobe, Japan) was used to measure the LBC. Immediately after it was obtained from the laboratory, the amniotic fluid in the EDTA tubes was analyzed. The LBC protocol has been described in detail in another publication [8]. Since lamellar bodies are similar in size to platelets, they can be counted in the platelet channel of the cell counter.
3.1. Measurement of L/S ratio in amniotic fluids
2.5. Measurement of the L/S ratio using LC–MS/MS and MALDI-TOF Amniotic fluid in the EDTA tube was centrifuged at 3600 rpm for 3 min at 4 ◦ C. In order to extract the phospholipids from the amniotic fluid, 0.2 mL centrifuged amniotic fluid was vortex-mixed with 0.2 mL methanol and 0.4 mL chloroform. This mixture was then centrifuged briefly at room temperature to facilitate separation of the two layers and the upper phase solution was removed. The tipcolumn is a pipette-tip-containing strong cation exchange material
The mass spectra of PC and SM in the amniotic fluid are shown in Fig. 1. Assigned peaks (signal to noise ratio >10) represent the six PC peaks (PC30:0, PC32:1, PC32:0, PC43:2, PC34:1, PC36:4) and one SM peak (SM34:1) from all samples. The peak intensity of PC32:0 was greater than that of other PC peaks from mature samples. The peak intensity of SM34:1 was prominent in immature samples. Some peaks were accompanied by ions 2 amu lower, corresponding to homologous molecules with one additional double bond. In a previous study, the L/S ratio was calculated using nine PC peaks (30:0, 32:1, 32:0, 34:2, 34:1, 36:4, 36:3, 36:2, 36:1) and three SM peaks (34:1, 36:1, 42:2) [7]. However, a low intensity peak (PC 36:3, PC 36:2, PC 36:1, SM 36:1, SM42:2) was difficult to find in the immature samples. The L/S ratio by MS was calculated from the sum peak intensity of the six PC peaks, and that of SM34:1. The assay precision (CV; n = 10) was
Contents lists available at ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Prediction of fetal lung maturity using the lecithin/sphingomyelin (L/S) ratio analysis with a simplified sample preparation, using a commercial microtip-column combined with mass spectrometric analysis Ho-Seok Kwak a , Hee-Jung Chung a,∗ , Young Sik Choi b , Won-Ki Min c , So Young Jung d a Department of Laboratory Medicine, Cheil General Hospital & Women’s Healthcare Center, Dankook University College of Medicine, Seoul, Republic of Korea b Department of Chemistry, Inha University, Incheon, Republic of Korea c Department of Laboratory Medicine, University of Ulsan College of Medicine & Asan Medical Center, Seoul, Republic of Korea d Department of Surgery, Hallym University Kangnam Sacred Heart Hospital, Seoul, Republic of Korea
a r t i c l e
i n f o
Article history: Received 12 December 2014 Received in revised form 20 April 2015 Accepted 10 May 2015 Available online 14 May 2015 Keywords: Lecithin/sphingomyelin ratio Fetal lung maturity Cation-exchange resin ESI-MS/MS MALDI-TOF
a b s t r a c t Fetal lung maturity is estimated using the lecithin/sphingomyelin ratio (L/S ratio) in amniotic fluid and it is commonly measured with thin-layer chromatography (TLC). The TLC method is time consuming and technically difficult; however, it is widely used because there is no alternative. We evaluated a novel method for measuring the L/S ratio, which involves a tip-column with a cation-exchange resin and mass spectrometry. Phospholipids in the amniotic fluid were extracted using methanol and chloroform. Choline-containing phospholipids such as lecithin and sphingomyelin were purified by passing them through the tip-column. LC–MS/MS and MALDI-TOF were used to directly analyze the purified samples. The L/S ratio by mass spectrometry was calculated from the sum peak intensity of the six lecithin, and that of sphingomyelin 34:1. In 20 samples, the L/S ratio determined with TLC was significantly correlated with that obtained by LC–MS/MS and MALDI-TOF. There was a 100% concordance between the L/S ratio by TLC and that by LC–MS/MS (kappa value = 1.0). The concordance between the L/S ratio by TLC and that by MALDI-TOF was also 100% (kappa value = 1.0). Our method provides a faster, simpler, and more reliable assessment of fetal lung maturity. The L/S ratio measured by LC–MS/MS and MALDI-TOF offers a compelling alternative method to traditional TLC. © 2015 Published by Elsevier B.V.
1. Introduction Respiratory distress syndrome (RDS) is a major contributor to neonatal morbidity and mortality worldwide, affecting approximately 1% of all live births and 10% of all preterm infants [1]. RDS is the most common respiratory disorder of premature newborns. It is diagnosed in ∼70% of very low birth weight infants (501–1500 g) in the USA [2]. The preterm birth rate has risen in most developed
∗ Corresponding author at: Department of Laboratory Medicine, Cheil General Hospital & Women’s Healthcare, Center and Dankook University College of Medicine, 1-19 Mukjeong-dong, Jung-gu, Seoul 100-380, Republic of Korea. Tel.: +82 2 2000 7290; fax: +82 2 2000 7780. E-mail address: [email protected] (H.-J. Chung). http://dx.doi.org/10.1016/j.jchromb.2015.05.012 1570-0232/© 2015 Published by Elsevier B.V.
countries [2]. Despite the decreased use of the fetal lung maturity (FLM) test, it is still very important for planning deliveries in the preterm interval [3,4]. Through 2011, the most widely used FLM test was the TDx Fetal Lung Maturity II (Abbott Diagnostics, Il, USA). The inability to order the TDx Fetal Lung Maturity II test will likely increase the number of requests for the lecithin/sphingomyelin (L/S) ratio and the lamellar body count (LBC) [3]. The L/S ratio was the first established technique to estimate adequate fetal surfactant production [5]. Currently, the L/S ratio of amniotic fluid is most often measured using thin-layer chromatography (TLC). The TLC method is a one-dimensional procedure often performed using the Fetal-Tek 200 kit from Helena Laboratories [6]. However, it is both technically difficult and time-consuming.
82
H.-S. Kwak et al. / J. Chromatogr. B 993 (2015) 81–85
Previously, we proposed new methods to measure the L/S ratio including liquid chromatography tandem mass spectrometry (LC–MS/MS) and matrix-assisted laser desorption and ionization time of flight mass spectrometry (MALDI-TOF MS) [7]. The aim of this study was to assess the ability of these new methods to predict FLM and to compare their performance to that of conventional TLC. 2. Material and methods 2.1. Materials and reagents The Micro tip-column (packing material: strong cation) was purchased from Harvard Apparatus (Holliston, MA, USA) and 2,5dihydroxybenzoic acid (DHB) was obtained from Sigma–Aldrich (Louis, MO, USA). All other chemicals of analytical grade, or HPLC grade, were obtained from J.T. Baker (Phillipsburg, NJ, USA). The Fetal-Tek 200 kit was purchased from Helena Laboratories (Beaumont, TX, USA). 2.2. Sample collection The consecutive series of physician-ordered L/S ratio samples, collected during a one year period, were examined in Asan Medical Center (Seoul, Republic of Korea). FLM testing was ordered for women presenting with symptoms of preterm labor and for asymptomatic women presenting before scheduled cesarean sections. Amniotic fluid obtained by amniocentesis was collected in EDTA tubes and plain tubes from non-diabetic patients with gestational ages between 21 and 35 weeks. RDS symptoms were confirmed by review of the newborn records. After routine analysis, the samples were stored at −20 ◦ C until they were analyzed by mass spectrometry (MS). 2.3. Measurement of L/S ratio by TLC We routinely performed the Fetal-Tek 200 procedure according to the manufacturer’s instructions, without any modifications. The amniotic fluid in the plain tube was extracted with a mixture of chloroform and methanol and applied across the channel of a TLC plate. The mobile phase is a mixture of chloroform, methanol, 2-propanol, triethylamine, and water. The lecithin and sphingomyelin bands were well separated from the other phospholipids and neutral lipids. In order to be visualized, plates were prepared with phosphoric acid containing a cupric acetate catalyst. The lecithin-to-sphingomyelin ratio was calculated by scanning the lecithin and sphingomyelin bands on the TLC plates.
with a sulphopropyl group. In order to equilibrate and rinse the material in the tip, 250 uL of 90% acetonitrile (ACN) with 0.1% formic acid was added to the top of the tip and then was dispensed the fluid by syringe. Next, 250 uL of extracted solution (lower phase solution) was added to the top of the tip and then slowly dispensed the fluid. To elute the phospholipids, 150 uL 90% ACN with 0.1% formic acid was added to the top of the tip and then slowly dispensed the fluid. In the LC–MS/MS analysis, samples were quantified by direct flow injection. The flow solvent (90% ACN with 0.1% formic acid) was added at a constant flow of 50 uL/min by the PerkinElmer series 200. The triple quadrupole mass spectrometry (API 2000, MDX/Sciex, Canada) was operated using the following settings: capillary voltage, +5.5 kV; temperature in ion source, 300 ◦ C; collision energy, 40; collision gas, nitrogen at a pressure of 1.5 10–5 Torr. A precursor ion scan of m/z 184.1 specific for phosphocholinecontaining lipids was used for quantification [9]. Data analysis was performed using the Analyst software. In the MALDI-TOF MS analysis, matrix stock solutions were made up in 80% methanol at a concentration of 10 mg/mL. A 1 uL aliquot of each sample solution was mixed with 10 uL of a matrix solution. A 1 uL aliquot of the final solution of sample and matrix was manually deposited onto a stainless steel plate. The sample plate was rapidly dried under a warm air stream to improve the homogeneity of crystallization. The MALDI-TOF mass spectra were acquired on a Voyager DE-STR workstation (PerSeptive Biosystems, Framingham, MA). This system utilizes a pulsed nitrogen laser, emitting 337 nm at 3 Hz. The MALDI-TOF was operated using the following settings: accelerating voltage, +20 kV; grid voltage, 60%; extraction delay time, 100 n sec; low mass gate, 400 Da. The “low mass gate” was turned on to prevent the detector from becoming saturated by ions from the matrix. For each mass spectrum, 150 single laser shots were averaged. In order to enhance the spectral resolution, all spectra were recorded in the reflector mode. Data analysis was performed using Data Explorer TM. 2.6. Statistical analysis Data were analyzed using the SPSS 18 statistical software (SPSS Inc., Chicago, IL, USA). The relationship among the various parameters was tested and expressed using Pearson’s correlation coefficient. P-values < 0.05 were considered statistically significant. Cohen’s Kappa was used to assess agreement between each method’s measure of fetal lung maturity (agreement: 0.6, substantial to almost perfect) [10].
2.4. Measurement of the lamellar body count (LBC)
3. Results and discussion
The Sysmex XE-2100 (Sysmex, Kobe, Japan) was used to measure the LBC. Immediately after it was obtained from the laboratory, the amniotic fluid in the EDTA tubes was analyzed. The LBC protocol has been described in detail in another publication [8]. Since lamellar bodies are similar in size to platelets, they can be counted in the platelet channel of the cell counter.
3.1. Measurement of L/S ratio in amniotic fluids
2.5. Measurement of the L/S ratio using LC–MS/MS and MALDI-TOF Amniotic fluid in the EDTA tube was centrifuged at 3600 rpm for 3 min at 4 ◦ C. In order to extract the phospholipids from the amniotic fluid, 0.2 mL centrifuged amniotic fluid was vortex-mixed with 0.2 mL methanol and 0.4 mL chloroform. This mixture was then centrifuged briefly at room temperature to facilitate separation of the two layers and the upper phase solution was removed. The tipcolumn is a pipette-tip-containing strong cation exchange material
The mass spectra of PC and SM in the amniotic fluid are shown in Fig. 1. Assigned peaks (signal to noise ratio >10) represent the six PC peaks (PC30:0, PC32:1, PC32:0, PC43:2, PC34:1, PC36:4) and one SM peak (SM34:1) from all samples. The peak intensity of PC32:0 was greater than that of other PC peaks from mature samples. The peak intensity of SM34:1 was prominent in immature samples. Some peaks were accompanied by ions 2 amu lower, corresponding to homologous molecules with one additional double bond. In a previous study, the L/S ratio was calculated using nine PC peaks (30:0, 32:1, 32:0, 34:2, 34:1, 36:4, 36:3, 36:2, 36:1) and three SM peaks (34:1, 36:1, 42:2) [7]. However, a low intensity peak (PC 36:3, PC 36:2, PC 36:1, SM 36:1, SM42:2) was difficult to find in the immature samples. The L/S ratio by MS was calculated from the sum peak intensity of the six PC peaks, and that of SM34:1. The assay precision (CV; n = 10) was
