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Quantification of phosphatidylserine, phosphatidic acid and free

Journal of Pharmaceutical and Biomedical Analysis42(2006)

506–512

Short communication

Quanti?cation of phosphatidylserine,phosphatidic acid and free

fatty acids in an ultrasound contrast agent by normal-phase

high-performance liquid chromatography with

evaporative light scattering detection

Erlend Hvattum a,Steinar Uran a,

Anne Gunvor Sandb?k a,1,Anders?A.Karlsson b,2,Tore Skotland a,?

a GE Healthcare Medical Diagnostics,Nycoveien2,N-0401Oslo,Norway

b GE Healthcare Medical Diagnostics,Per Albin Hanssons vag41,S-20512Malm?,Sweden

Received18November2005;received in revised form25April2006;accepted25April2006

Available online9June2006

Abstract

Sonazoid TM is a new contrast agent for ultrasound imaging.The product is an aqueous suspension of per?uorobutane microbubbles coated with phospholipids obtained from hydrogenated egg phosphatidylserine(H-EPS).A normal-phase high-performance liquid chromatographic(HPLC) method with evaporative light scattering detection was developed for quanti?cation of free fatty acids,phosphatidylserine and phosphatidic acid in H-EPS and Sonazoid TM.Separation of the lipids was carried out on an HPLC diol column and a gradient of chloroform and methanol with 0.2%formic acid titrated to pH7.5with ammonia.The calibration standards contained stearic acid,distearoyl-phosphatidic acid(DSPA)and distearoyl-phosphatidylserine(DSPS)in the concentration range of0.016–1.0mg/ml(0.4–25?g injected).The method was validated with a limit of quanti?cation of the three lipids set to0.4?g(approximately20–60?M).The best?t of the three calibration curves were obtained when the logarithmic transformed theoretical lipid concentration was plotted against the logarithmic transformed area under the peak and?tted to a second order polynomial equation.Stearic acid,DSPA and DSPS were analysed with an intermediate precision ranging from4.4%to5.3%R.S.D.and they were extracted from an aqueous suspension with a recovery ranging from103.3%to113.3%.The sum of total phospholipid concentration determined in H-EPS ranged from96.4%to103.2%of the theoretical values.The lipids in the ultrasound product were quantitated with a repeatability ranging from6.2%to11.7%R.S.D.

Keywords:HPLC;Evaporative light scattering detector;Phosphatidylserine;Phosphatidic acid;Free fatty acids

1.Introduction

Phospholipids are the main structural and functional com-pounds of cellular membranes and due to their emulsifying properties they are used commercially in different type of prod-ucts.Phospholipid vesicles,i.e.liposomes,have become impor-

?Corresponding author.Tel.:+4723185666;fax:+4723186008.

E-mail address:Tore.Skotland@https://www.wendangku.net/doc/ba589345.html,(T.Skotland).

1Present address:AGS,Nycomed Pharma AS,Solb?rveien25,N-2418 Elverum,Norway.

2Present address:A?AK,Novo Nordisk AS,Novo Nordisk Park,DK-2760 M?a l?v,Denmark.tant as drug delivery systems in general and as drug targeting applications in particular.Liposomes can be made from a vari-ety of materials,which make them versatile as drug carrier systems.

With the increasing use of liposomes as drug carriers it is important to have proper analytical techniques to quanti-tate and characterise the composition of the liposomes and their raw material.Previous quanti?cation of phospholipids has been obtained with thin-layer chromatography[1].In recent years,application of high-performance liquid chromatography (HPLC)has become more important and numerous HPLC methods have been described for the separation of phospho-lipids(for review,see[2]).Separation of the different phos-

E.Hvattum et al./Journal of Pharmaceutical and Biomedical Analysis42(2006)506–512507

pholipid classes is obtained with normal-phase chromatogra-phy with silica as the most frequently used stationary phase [3–7].In addition,modi?ed silica,particularly diol[8–12], cyanopropyl[13,14]and aminopropyl[15–17]have also been used.With respect to the mobile phase different solvent mix-tures have been used;n-hexane–2-propanol–water/acids/bases [8,11,17],acetonitrile–methanol–water/acids/bases[11,15,16] or chloroform–methanol–ammonium hydroxide[4–6,12].

The major dif?culty in phospholipid analysis has been the detection of the substance.Only phospholipids with unsaturated fatty acids(one or more double bonds)have some absorption in the low UV range,i.e.at or below210nm.The problem with UV detection is that the response depends on the degree of unsaturation of the phospholipid fatty acid chain and makes the choice of standards dif?cult.In addition,this detection method requires solvents with high spectral transparency.Other detec-tion principles that may be used are?uorescence detection(by post-column formation of mixed micelles)[18],mass spectrom-etry(MS)[19,20],?ame ionisation detection(FID)[21,22]and refractive index detection(RI)[23–25].MS represents a very sensitive detection method that is very useful for qualitative or semiquantitative analyses of phospholipids;it is however,more complicated to use this detector for exact quantitative analyses of mixtures of phospholipids/lipids[19,20].FID is not at present commercially available in combination with HPLC and RI is not compatible with gradient elution.Evaporative light scattering (ELS)detection,on the other hand,enables gradient elution and can be used with all volatile solvents as mobile phases.The ELS detector response is primarily caused by the mass of the analyte [26,27]and has a reasonable sensitivity as phospholipids have been reported to be quantitated down to0.1?g[5,28].

A new contrast agent for ultrasound imaging has been devel-oped by GE Healthcare.This ultrasound product(Sonazoid TM) is an aqueous suspension of per?uorobutane(PFB)microbub-bles coated with phospholipids obtained from hydrogenated egg phosphatidylserine(H-EPS).The main phospholipids in H-EPS are phosphatidylserine(PS)and phosphatidic acid(PA).

We have earlier presented a normal-phase HPLC method using a narrow-bore diol column with mass spectrometric detec-tion for speci?c quanti?cation of one molecular species of PS from human blood,i.e.palmitoyl-stearoyl-phosphatidylserine [29].The main objective of the present work was to develop a normal-phase HPLC method with ELS detection for the sepa-ration and quanti?cation of both phospholipids and breakdown products of phospholipids in H-EPS and Sonazoid TM.The sep-aration of free fatty acids(FFA),PA and PS were optimised and the three lipid classes were quantitated using external standard curves.In addition,the lysoforms of PA and PS were separated from their parent phospholipid and determined qualitatively.The validation of the method is presented in this paper.To our knowl-edge this is the?rst paper describing a quantitative method for analysis of the lipid content of an ultrasound contrast agent.We show that by using this method it is possible to obtain good quantitative data of these lipid classes in a short runtime(20min only)with only one species from each lipid class as standard. The method would be expected to be useful also for analysis of other lipid mixtures,e.g.liposomes.2.Experimental

2.1.Materials

Sonazoid TM was from GE Healthcare Bio-Sciences,Oslo, Norway.Chloroform(stabilised with amylene(2-methyl-2-butene))was either LiChrosolv grade from Merck or HiPersolv grade from BdH.Methanol was LiChrosolv grade from Merck. Formic acid(98–100%),ammonia(25%)and hydrochloric acid were pro analysis grade from Merck.Stearic acid and palmitic acid were from Sigma Chemical Company,St.Louis,MO, USA.1,2-Dipalmitoyl-sn-glycero-3-phosphate(DPPA),1,2-distearoyl-sn-glycero-3-phosphate(DSPA),1,2-dipalmitoyl-sn-glycero-3-[phospho-l-serine](DPPS),1,2-distearoyl-sn-glycero-3-[phospho-l-serine](DSPS),1-stearoyl-2-hydroxy-sn-glycero-3-phosphatidic acid(lyso-PA),hydrogenated plant l-?-phosphatidylinositol(PI)were from Avanti Polar Lipids Inc.,Alabaster,Alabama,USA.Monoacyl-sn-glycero-3-phospho-l-serine(lyso-PS)containing primarily octadecanoic acid,sphingomyelin(Sm),l-?-lysophosphatidylethanolamine palmitoyl(lyso-PE)and l-?-lysophosphatidylcholine palmitoyl (lyso-PC)were from Sigma Chemical Company.1,2-Distearoyl-sn-glycero-3-phosphoethanolamine(DSPE)and1,2-distearoyl-sn-glycero-3-phosphocholine(DSPC)were from Sygena Inc., Cambridge,MA,USA.The purity of the standard compounds was approximately99%.Hydrogenated egg phosphatidylserine (H-EPS)was from NOF Corporation,Amagasaki-Shi,Hyogo, Japan.

2.2.Chromatographic conditions

The chromatographic method is based on a method devel-oped in our laboratory for LC–MS analyses[29].The chro-matographic system consisted of a Spectra-Physics SP8800 pump connected to a Spectra-Physics SP8880autosampler, equipped with a100?l sample loop(Rheodyne).A Sedex55 ELS detector,SEDERE,Alfortville,France,was used for detec-tion of the lipids.The lipids were separated on a LiChroCART, LiChrospher100Diol,250mm×4mm(5?m)column(Merck) with a LiChrospher100Diol,4mm×4mm(5?m)precolumn (Merck).The mobile phase consisted of chloroform(mobile phase A)and methanol with0.2%(v/v)formic acid titrated to pH7.5(if not otherwise stated)with ammonia(mobile phase B). The lipids in the samples were separated by running a gradient starting at100%mobile phase A,decreasing to64%A in9min, and further decreasing to40%A in4min and then back to100% A in4min.Total run time for each sample was set to30min and the?ow rate was1.3ml/min.The analyses were performed at ambient temperature.The samples were kept at room tempera-ture and25?l was injected for each analysis.One injection per vial was performed.The ELS detector drift-tube temperature was set to40?C,the nitrogen gas pressure was set to2.0bar and gain was set to6if not otherwise stated.

2.3.Preparation of standards

The calibration standards were prepared by dissolving approximately equal amounts of stearic acid,DSPA and DSPS

508 E.Hvattum et al./Journal of Pharmaceutical and Biomedical Analysis42(2006)506–512

in chloroform/methanol(75:25,v/v)and further dilution of the standards to the following concentrations(in mg/ml):0.016, 0.031,0.063,0.13,0.25,0.50and1.0of stearic acid;0.016, 0.032,0.064,0.13,0.25,0.51and1.0of DSPA;and0.016,0.031, 0.062,0.12,0.25,0.50and1.0of DSPS.The calibration stan-dards were then distributed to HPLC-vials,dried by evaporation under nitrogen and kept at?20?C.Prior to analysis the stan-dards were redissolved in chloroform/methanol/water(65:25:4, v/v/v).

The control samples containing stearic acid,DSPA,DSPS, lyso-PS and lyso-PA(approximately0.2mg/ml)and other stan-dard lipid solutions were prepared similarly to the calibration standards.

2.4.Lipid extraction of drug product

Drug product samples were prepared by reconstitution of the ultrasound drug product in sterile water and dilution in sucrose (92mg/ml).Immediately after reconstitution,the lipids in the product were extracted essentially as described by J¨a¨a skel¨a inen [16].Brie?y,2ml of reconstituted Sonazoid TM was transferred to a separation funnel.The lipids in the product were extracted by adding7.5ml of methanol/chloroform(2:1,v/v),mixed thor-oughly,followed by2.5ml of0.1M hydrochloric acid and2.5ml of chloroform.The solution was mixed thoroughly before the lower chloroform phase was sampled.The extraction proce-dure was repeated once and the pooled chloroform phase was evaporated to dryness under a stream of nitrogen gas.The residues were dissolved in500?l of chloroform/methanol/water (65:25:4,v/v/v).

2.5.Sample analysis and validation parameters

The samples were analysed in sequences together with cal-ibration standards,control samples and injection blanks.The calibration standards were positioned at the beginning and at the end of each sequence,while the control samples and the injection blanks were randomly placed in the sequence together with the samples.

The three standard curves were evaluated from three calibra-tion graphs prepared and run on three different days.Repeata-bility was evaluated by analysing six or seven sample replicates at medium and high concentration of stearic acid,DSPA and DSPS.Intermediate precision was evaluated by analysing three sample replicates of the control sample on three different days. The ef?ciency of the extraction was examined in samples pre-pared by dissolving a known concentration of DSPA and DSPS in a10%(w/v)sucrose solution.The solution was lyophilised and stearic acid was added.The dry mixture was dissolved in 2ml water prior to extraction and analysis as described.

The repeatability and accuracy of analysing lipids in H-EPS were evaluated by analysing three different batches of approximately0.5mg/ml of H-EPS dissolved in chlo-roform/methanol/water(65:25:4,v/v/v).The repeatability of analysing lipids in Sonazoid TM was evaluated by extracting10 vials of lyophilised Sonazoid TM from1batch.2.6.Data handling

PE Nelson ACCESS*CHROM GC/LC data sampling sys-tem,v.1.9was used for sampling and integration of the chro-matograms.GraphPad TM Prism v.2.0was used for regression analysis and for calculating the sample concentration.Microsoft Excel,v.5.0was used for statistical calculation.

3.Results and discussion

3.1.Choice of calibration standards

H-EPS is composed of85–90%(w/w)of PS and10–15% (w/w)of PA.The fatty acid composition of the phospholipids (calculated as w/w)is approximately30%of palmitic acid (C16:0),60%of stearic acid(C18:0),5%of arachidic acid (C20:0)and5%of behenic acid(C22:0).The species distri-bution of PS and PA in H-EPS was determined by negative elec-trospray ionisation(ESI)tandem quadrupole mass spectrome-try(MS/MS)essentially as previously described[30].Brie?y, negative ESI-MS of H-EPS yielded mass spectra,which dis-played deprotonated molecules representing the various species of PS and PA in H-EPS.The identity con?rmation of the species was obtained by MS/MS experiments were the depro-tonated molecules were selected by MS1and fragmented by collision-induced dissociation.The resulting product ion spec-tra displayed carboxylate anion fragments,which identi?ed the PS and PA species in H-EPS.Calculated from the intensi-ties of the deprotonated molecules,the MS data showed that the main molecular species of PS were palmitoyl-stearoyl-phosphatidylserine(PSPS),accounting for approximately60% (w/w),and distearoyl-phosphatidylserine(DSPS),accounting for approximately30%(w/w).Corresponding molecular species were also found in PA.For calibration it was decided to use pure lipid standards for each lipid class to represent this mixture,i.e. stearic acid,DSPA and DSPS.

3.2.Method development and optimisation of the mobile phase

Initially,mobile phase B consisted of a mixture of methanol and1.25%(v/v)ammonia,giving a relative rapid deterioration of the column due to the high pH.The pH of the mobile phase B was then adjusted to7.5with formic acid,which was more suitable for the column and also increased the ELS detector response for PA and PS.The maximum amount of formic acid and ammonia that could be added to the methanol was0.2% (v/v)formic acid adjusted to pH7.5with ammonia.At higher ammonium formate concentration the HPLC system was repeat-edly blocked when running the gradient,probably because the chloroform precipitated the ammonium formate above a criti-cal concentration.With a pH below7.5in mobile phase B the fatty acid peak was split in two(not shown).This is probably due to a gradual change in polarity of the fatty acid due to a higher degree of protonation at lower pH.Mobile phase B at pH 7.5was therefore routinely used for all analysis.Previously,we have used similar chromatographic conditions with the mobile

E.Hvattum et al./Journal of Pharmaceutical and Biomedical Analysis42(2006)506–512509

phase at pH5.3for quanti?cation of PSPS in human blood using MS detection[29].

3.3.Optimisation of the detector temperature

The ELS detector response is dependent on the temperature of the drift-tube[31].When using the present method the response of the lipids gradually decreased at a temperature increment from 40to90?C.When the drift-tube temperature was increased from 40to60?C,a two-to three-fold decrease in the response was observed.A further temperature increase of10?C led to an even more dramatically drop in response,as only approximately5% of the response at40?C was observed at70?C.From70to 90?C only small changes in the response was observed.Thus a drift-tube temperature of40?C was chosen.A similar drift-tube temperature has previously been reported for determination of phospholipids with HPLC and ELS detection[32,33],while others have reported an optimal drift-tube temperature of85?C [34].The most favourable drift-tube temperature of the ELS detector for phospholipid determination will most probably vary depending on the make of the ELS detector.

3.4.Separation and speci?city

Separation of up to nine lipid classes was achieved by the HPLC method(Fig.1).As shown in Fig.1A,PE and Sm co-eluted with mobile phase at pH7.5,but it was possible to obtain some separation between the two lipids by reducing the pH of mobile phase B to5.3(Fig.1B).When each phospholipid class was analysed separately,Sm eluted as two peaks(not shown) as reported by others[4–6,32].Fig.2A shows that FFA,PA and PS were well separated with a resolution factor between DSPS and DSPA of;R S=2.94±0.03(mean±S.D.,n=3).Fig.2B shows that there was little difference in retention times when comparing the pure calibration standards with PA and PS from H-EPS.The two lysophospholipids,lyso-PS and lyso-PA,eluted essentially as one single peak(Fig.2A).Since the lysophospho-lipids were only qualitatively determined,no attempt was made in improving the separation between lyso-PS and lyso-PA.

3.5.Limit of quanti?cation

According to published recommendations,the limit of quan-ti?cation(LOQ)of a method can be set to a speci?c con-centration provided that the repeatability of analysing at this concentration is below20%relative standard deviation(R.S.D.) of the mean[35].Based on the prevalidation work,the lowest calibration standard at0.016mg/ml(corresponding to0.4?g of lipid injected)was chosen as the LOQ of the method.Stearic acid and DSPS were analysed at0.4?g with a repeatability of6.2%R.S.D.(n=7)and10.4%R.S.D.(n=7),respectively. The response of DSPA was slightly poorer than the two other lipids and the LOQ for DSPA was therefore at?rst set to0.8?g, but with time DSPA was repeatedly detected at0.4?g with a repeatability ranging from11.2%to14.1%R.S.D.(three inde-pendent analyses with two sample replicates for each analysis). The lipids have successfully been detected at lower

concentra-Fig.1.(A)Typical chromatogram of a mixture of phospholipids analysed with mobile phase B at pH7.5.Peaks:(1)unknown;(2)palmitic acid (0.25mg/ml);(3)DSPC(0.13mg/ml);(4)DSPE and Sm co-chromatographing (0.13mg/ml of each);(5)lyso-PC(0.13mg/ml);(6)DPPA(0.25mg/ml);(7) lyso-PE(0.13mg/ml);(8)PI and peak from chloroform co-chromatographing (0.13mg/ml);(10)DPPS(0.25mg/ml);(11)lyso-PS(0.22mg/ml).(B)Typi-cal chromatogram of a mixture of phospholipids analysed with mobile phase

B at pH5.3.Peaks:(1)unknown;(2)palmitic acid(0.25mg/ml);(3)DSPC

(0.13mg/ml);(4)DSPE(0.13mg/ml);(5)Sm(0.13mg/ml);(6)DPPA and lyso-PC co-chromatographing(0.25and0.13mg/ml);(7)lyso-PE(0.13mg/ml);(8) PI(0.13mg/ml);(9)peak from chloroform;(10)DPPS(0.25mg/ml);(11)lyso-PS(0.22mg/ml).Conditions are described in the

text.

Fig.2.(A)Typical chromatogram of a standard mixture of lipids.Peaks:(1) stearic acid(0.21mg/ml);(2)DSPA(0.20mg/ml);(3)peak from mobile phase;

(4)DSPS(0.21mg/ml);(5)lyso-PS(0.21mg/ml)and lyso-PA(0.20mg/ml).

(B)Typical chromatogram of H-EPS(1.0mg/ml).Peaks:(2)PA and(4)PS. Conditions are described in the text.

510 E.Hvattum et al./Journal of Pharmaceutical and Biomedical Analysis42(2006)506–512

tions than0.4?g,e.g.stearic acid has been detected at0.125?g, but with varying response.Detection limits for different phos-pholipids after analysis with ELS detection have previously been reported to range between0.25and0.5?g[28,36]and even as low as0.1?g[5].In addition,it has been found that neutral lipids elicit greater ELS responses than polar lipids[37].

The LOQ of an HPLC-method using the present type of ELS detector can be improved by increasing the ELS detector gain. Preliminary experiments showed that it was possible to increase the gain to8–9without a concomitant increase of the noise and thereby theoretically reducing the LOQ of the method(not shown).The LOQ of lyso-PS and lyso-PA was not determined but was approximately three times higher than for stearic acid, DSPA and DSPS(not shown).Generally,the detector response of the two lysophospholipids were much poorer compared to the other three lipids(Fig.2).

3.6.Calibration

The concentration of the calibration standards ranged from 0.016to1.0mg/ml for stearic acid and DSPS and,initially,from 0.032to1.0mg/ml for DSPA.When the theoretical concentra-tions of the lipids were plotted against the calculated peak areas, the curve appearance was sigmoidal for DSPA and DSPS and exponential for stearic acid(Fig.3A and B).Similar response curves have previously been reported for this type of detector [5,6,36,38].This is believed to be due to the dependence of the light scattering mechanisms on the size of the particles formed by the non-volatile compounds and the particle size increases with increasing concentration of the compounds[27,39].

Due to the sigmoidal and exponential appearance of the three curves,the best?t of the calibration curves were obtained when the logarithmic transformed theoretical lipid concentration was plotted against the logarithmic transformed area under the peak. The linearity of a calibration curve can be described by the equa-tion;y=a+bx m[40].The m values for the three transformed curves of stearic acid,DSPA and DSPS were:m=1.25±0.20 (mean±S.D.,n=3),m=0.012±0.001(mean±S.D.,n=3) and m=0.011±0.001(mean±S.D.,n=3),respectively.In addition,an F-test to evaluate linear versus quadratic regres-sion was performed.For DSPS and DSPA,the values from the F-test ranged from116to2085,clearly showing that non-linear regression?ts the data signi?cantly better than linear regres-sion.For stearic acid,the values from the F-test were0.77,9.9 and32.Even if the results from the F-test are not conclusive, they indicate that non-linear regression?ts the data better in two of three curves.In view of this it was decided to?t the three transformed calibration curves to a second-order polyno-mial equation:y=a+bx+cx2(Fig.3C).

The estimated parameters of the calibration curves from three series of analysis are listed in Table1and shown to be repro-ducible.The reverse predicted standard points showed a devi-ation ranging from?3%to6%of the nominal concentrations over the whole range of the three standard curves.This was reproducible in three analytical series(not shown)and shows that the calibration model chosen for the three curves gives a good?t of the standard

points.Fig.3.Typical response curves where each point on the curves represent the mean of two parallels.(A)Response curves of DSPA( )and DSPS(?)and (B)response curve of stearic acid.The curves were?tted to a cubic spline equa-tion.(C)Calibration curves of stearic acid( ),DSPA( )and DSPS(?).The logarithmic transformed concentration of lipids were plotted against the loga-rithmic transformed area under the peak and?tted to a second order polynomial equation.

3.7.Repeatability,intermediate precision and accuracy

The repeatability and intermediate precision of the method were found to range from0.9%to3.3%R.S.D.and4.4% to 5.3%R.S.D.(Table2),respectively.The ef?ciency of extraction was determined as described in Section2and found to be(in%of theoretical concentration):113.3±5.8 (mean±S.D.,n=3),110.8±4.1(mean±S.D.,n=6)and

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511

Table 1

Parameters of the calibration curves Calibration curves a

r 2

Stearic acid 6.40±0.03 1.32±0.050.07±0.050.9990±0.0004DSPA 7.16±0.020.69±0.16?0.85±0.080.9984±0.0013DSPS

7.19±0.03

0.71±0.02

?0.66±0.06

0.9986±0.0008

For the three standard curves the logarithmic transformed theoretical lipid concentration was plotted against the logarithmic transformed area under the peak and ?tted to the equation:y =a +bx +cx 2.The regression parameters were estimated after analysing three calibration series with two parallels for each standard.The values are the mean ±S.D.(n =3).

Table 2

The repeatability and intermediate precision of the method

Stearic acid

DSPA

DSPS

Mean concentration (mg/ml)

R.S.D.(%)Mean concentration (mg/ml)R.S.D.(%)Mean concentration (mg/ml)R.S.D.(%)Repeatability n =6a 0.129±0.003

2.0

0.133±0.003 2.40.130±0.003 2.3n =7b 0.982±0.025 2.60.935±0.0080.90.979±0.032 3.3Intermediate precision n =9c 0.226±0.012

5.3

0.213±0.011

5.0

0.235±0.010

4.4

For the repeatability the values are expressed as the mean ±S.D.For the intermediate precision the values are expressed as the grand average of three series of analysis each with three sample replicates ±S.D.

a Theoretical concentrations:0.126mg/ml (stearic acid);0.127mg/ml (DSPA);0.125mg/ml (DSPS).

b Theoretical concentrations:1.006mg/ml (steari

c acid);1.016mg/ml (DSPA);0.998mg/ml (DSPS).c Theoretical concentrations:0.21mg/ml (stearic acid);0.20mg/ml (DSPA);0.22mg/ml (DSPS).

103.3±2.1(mean ±S.D.,n =6)for stearic acid,DSPA and DSPS,respectively.3.8.Stability

The stability of stearic acid,DSPA and DSPS in chloro-form/methanol/water (65:25:4,v/v/v)at both medium and high concentration,i.e.0.13and 1.0mg/ml,respectively,was exam-ined with the samples kept at room temperature.After 5days,the mean percentage of the 0-time value at medium and high con-centration for stearic acid,DSPA and DSPS were found to be (n =3):98.5±4.5and 101.9±0.0;101.4±4.4and 113.9±6.2;101.1±0.0and 102.9±0.0,respectively.Thus these lipids were stable for at least 5days at room temperature.Correspond-

ingly,the stability of the phospholipids after extraction was also examined.Extracted DSPA and DSPS redissolved in chloro-form/methanol/water (65:25:4,v/v/v)were found to be stable for at least 7days at room temperature (not shown).3.9.Repeatability and accuracy of quantitating lipids in H-EPS and Sonazoid TM

Table 3shows that the accuracy of estimating total phospho-lipids (PA and PS)in three H-EPS batches ranged from 96.4%to 103.2%.This indicates that DSPA and DSPS are useful calibra-tion standards for quanti?cation of PA and PS in H-EPS.Table 3further shows that the repeatability of analysing PA and PS in H-EPS was below 2%R.S.D.

Table 3

Quantitation of free fatty acids (FFA),phosphatidic acid (PA)and phosphatidylserine (PS)in H-EPS and Sonazoid TM

FFA

Concentration as %of theoretical value

Mean concentration (mg/ml)

R.S.D.(%)

Mean concentration (mg/ml)R.S.D.(%)

Batch of H-EPS 1(n =3)a

0.075±0.001 1.30.426±0.005 1.296.4±1.02(n =3)b

Sonazoid TM

n =10

0.024±0.0028

11.70.039±0.0028

7.2

0.181±0.011

6.2

Three different batches of H-EPS were analysed.The values are the mean ±S.D.In addition,the sum of total phospholipid concentration as percentage (%)of theoretical value was calculated.Ten different glasses of Sonazoid TM from one batch were analysed.The values are the mean ±S.D.

a Theoretical concentration of H-EPS:0.52mg/ml.

b Theoretical concentration of H-EPS:0.46mg/ml.

c Theoretical concentration of H-EPS:0.59mg/ml.

512 E.Hvattum et al./Journal of Pharmaceutical and Biomedical Analysis42(2006)506–512

The repeatability of lipid analysis in Sonazoid TM was found to be in the range of6.2–11.7%R.S.D.(Table3).Based on the previous precision data(Tables2and3)these results indicate that in addition to the variations of the analysis there is also some variation in the glass to glass content of lipids in the Sonazoid TM product.

In conclusion,the present method was developed for quan-ti?cation of FFA,PA and PS in H-EPS and in the ultrasound contrast agent Sonazoid TM.The method has successfully been validated and the method provides a suf?ciently sensitive,accu-rate and reproducible analytical procedure for analysis of these lipids in one run of20min with only one species from each lipid class as standard.The method is applicable for the anal-ysis of lipid constituent in both H-EPS and Sonazoid TM,and also for analysis of other lipid mixtures such as found in e.g. liposomes.

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关于时间管理的英语作文 manage time

How to manage time Time treats everyone fairly that we all have 24 hours per day. Some of us are capable to make good use of time while some find it hard to do so. Knowing how to manage them is essential in our life. Take myself as an example. When I was still a senior high student, I was fully occupied with my studies. Therefore, I hardly had spare time to have fun or develop my hobbies. But things were changed after I entered university. I got more free time than ever before. But ironically, I found it difficult to adjust this kind of brand-new school life and there was no such thing called time management on my mind. It was not until the second year that I realized I had wasted my whole year doing nothing. I could have taken up a Spanish course. I could have read ten books about the stories of successful people. I could have applied for a part-time job to earn some working experiences. B ut I didn’t spend my time on any of them. I felt guilty whenever I looked back to the moments that I just sat around doing nothing. It’s said that better late than never. At least I had the consciousness that I should stop wasting my time. Making up my mind is the first step for me to learn to manage my time. Next, I wrote a timetable, setting some targets that I had to finish each day. For instance, on Monday, I must read two pieces of news and review all the lessons that I have learnt on that day. By the way, the daily plan that I made was flexible. If there’s something unexpected that I had to finish first, I would reduce the time for resting or delay my target to the next day. Also, I would try to achieve those targets ahead of time that I planed so that I could reserve some more time to relax or do something out of my plan. At the beginning, it’s kind of difficult to s tick to the plan. But as time went by, having a plan for time in advance became a part of my life. At the same time, I gradually became a well-organized person. Now I’ve grasped the time management skill and I’m able to use my time efficiently.

英语演讲稿：未来的工作

英语演讲稿：未来的工作这篇《英语演讲稿范文：未来的工作》，是特地，希望对大家有所帮助！热门演讲推荐：竞聘演讲稿 | 国旗下演讲稿 | 英语演讲稿 | 师德师风演讲稿 | 年会主持词 | 领导致辞 everybody good afternoon:. first of all thank the teacher gave me a story in my own future ideal job. everyone has a dream job. my dream is to bee a boss, own a pany. in order to achieve my dreams, i need to find a good job, to accumulate some experience and wealth, it is the necessary things of course, in the school good achievement and rich knowledge is also very important. good achievement and rich experience can let me work to make the right choice, have more opportunities and achievements. at the same time, munication is very important, because it determines whether my pany has a good future development. so i need to exercise their municative ability. i need to use all of the free time to learn

Quantification of Fetal DNA by Use of Methylation-Based DNA Discrimination

Quantification of Fetal DNA by Use of Methylation-Based DNA Discrimination Anders O.H.Nygren,1Jarrod Dean,1Taylor J.Jensen,1Selena Kruse,1William Kwong,1 Dirk van den Boom,2and Mathias Ehrich2* BACKGROUND:Detection of circulating cell-free fetal nucleic acids in maternal plasma has been used in non-invasive prenatal diagnostics.Most applications rely on the qualitative detection of fetal nucleic acids to deter-mine the genetic makeup of the fetus.This method leads to an analytic dilemma,because test results from samples that do not contain fetal DNA or are contam-inated with maternal cellular DNA can be misleading. We developed a multiplex approach to analyze regions that are hypermethylated in placenta relative to mater-nal blood to evaluate the fetal portion of circulating cell-free DNA isolated from maternal plasma. METHODS:The assay used methylation-sensitive re-striction enzymes to eliminate the maternal(unmeth-ylated)fraction of the DNA sample.The undigested fetal DNA fraction was then coamplified in the pres-ence of a synthetic oligonucleotide to permit com-petitive PCR.The amplification products were quantified by single-base extension and MALDI-TOF MS analysis. RESULTS:Using2independent markers,(sex determin-ing region Y)-box14(SOX14)and T-box3(TBX3),we measured a mean of151copies of fetal DNA/mL plasma and a mean fetal fraction of0.13in samples obtained from pregnant women.We investigated242 DNA samples isolated from plasma from pregnant and nonpregnant women and observed an analytical sensi-tivity and specificity for the assay of99%and100%, respectively. CONCLUSIONS:By investigating several regions in paral-lel,we reduced the measurement variance and enabled quantification of circulating cell-free DNA.Our results indicate that this multiplex methylation-based reac-tion detects and quantifies the amount of fetal DNA in a sample isolated from maternal plasma. ?2010American Association for Clinical Chemistry Since its discovery,circulating cell-free fetal DNA(ccff DNA)3isolated from maternal plasma(1)has drawn much attention because it provides genetic informa-tion about the fetus with a reduced risk of complica-tions compared with invasive procedures(2).Several applications for noninvasive prenatal diagnosis (NIPD)are available for the detection of fetal sex (3,4),rhesus D blood type(5,6),and paternal-derived mutations(7,8).Despite the achievements made in both qualitative and quantitative analysis of fetal DNA in maternal plasma(9),there are still limitations to this technology.Most applications rely on differences in DNA sequence between the fetal and maternal ge-nomes.In these applications,absence of the investi-gated fetal sequence would correspond to noninher-itance of the paternal allele.A lack of detectable signal, however,could also be due to a low fetal DNA concen-tration in the sample(10).Many factors can contribute to these false-negative results,but all can lead to false interpretation of the fetal DNA component.Therefore, a method that can be used to detect and quantify a universal fetal DNA marker is needed(11,12). Until recently the search for a universal fetal marker was largely restricted to genetic polymor-phisms located on the autosomal chromosomes(13), but to ensure at least1informative marker a more complex test is necessary that consumes a large portion of the available DNA sample.A potential alternative to sequence-based detection of fetal DNA is to use other factors to discriminate between maternal and fetal DNA(14–16).Recently several independent studies have presented a comprehensive set of genomic regions in which fetal-specific tissue is differentially methyl-ated compared with the corresponding maternal pe-ripheral blood mononuclear cells(PBMCs)(17–21). This epigenetic difference can be explored to establish an assay that targets the fetal portion of the cell-free DNA in plasma. 1Sequenom Center for Molecular Medicine,San Diego,CA;2Sequenom,San Diego,CA. *Address correspondence to this author at:3595John Hopkins Court,San Diego 92122,CA.Fax858-202-9084;e-mail MEhrich@https://www.wendangku.net/doc/ba589345.html,. Received February26,2010;accepted July20,2010.Previously published online at DOI:10.1373/clinchem.2010.146290 3Nonstandard abbreviations:ccff DNA,circulating cell-free fetal DNA;NIPD, noninvasive prenatal diagnostics;PBMC,peripheral blood mononuclear cell; FQA,fetal quantifier assay. Clinical Chemistry56:10 1627–1635(2010) Molecular Diagnostics and Genetics 1627

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关于坚持的英语演讲稿 Results are not important, but they can persist for many years as a commemoration of. Many years ago, as a result of habits and overeating formed one of obesity, as well as indicators of overall physical disorders, so that affects my work and life. In friends to encourage and supervise, the participated in the team Now considered to have been more than three years, neither the fine rain, regardless of winter heat, a day out with 5:00 time. The beginning, have been discouraged, suffering, and disappointment, but in the end of the urging of friends, to re-get up, stand on the playground. 成绩并不重要，但可以作为坚持多年晨跑的一个纪念。多年前，由于庸懒习惯和暴饮暴食，形成了一身的肥胖，以及体检指标的全盘失常，以致于影响到了我的工作和生活。在好友的鼓励和督促下，参加了晨跑队伍。现在算来，已经三年多了，无论天晴下雨，不管寒冬酷暑，每天五点准时起来出门晨跑。开始时，也曾气馁过、痛苦过、失望过，但最后都在好友们的催促下，重新爬起来，站到了操场上。 In fact, I did not build big, nor strong muscles, not a sport-born people. Over the past few years to adhere to it, because I have a team behind, the strength of a strongteam here, very grateful to our team, for a long time, we encourage each other, and with sweat, enjoying common health happy. For example, Friends of the several run in order to maintain order and unable to attend the 10，000 meters race, and they are always concerned about the brothers and promptly inform the place and time, gives us confidence and courage. At the same time, also came on their own inner desire and pursuit for a good health, who wrote many of their own log in order to refuel for their own, and inspiring. 其实我没有高大身材，也没健壮肌肉，天生不属于运动型的人。几年来能够坚持下来，因为我的背后有一个团队，有着强大团队的力量，在这里，非常感谢我们的晨跑队，长期以来，我们相互鼓励着，一起流汗，共同享受着健康带来的快

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关于工作的优秀英语演讲稿

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