Voltammetry and quantification of the contraceptive drug norethisterone

Voltammetry and quantification of the contraceptive drug norethisterone
Voltammetry and quantification of the contraceptive drug norethisterone

Journal of Pharmaceutical and Biomedical Analysis 43(2007)

499–505

V oltammetry and quanti?cation of the contraceptive drug norethisterone

in bulk form and pharmaceutical formulation

M.M.Ghoneim a ,?,A.M.Abushoffa b ,Y .I.Moharram a ,H.S.El-Desoky a

a

Chemistry Department,Faculty of Science,Tanta University,31527Tanta,Egypt

b

Department of Pharmaceutical Chemistry,Faculty of Pharmacy,Alfateh University,P .O.Box 13620,Tripoli,Libya

Received 26March 2006;received in revised form 26July 2006;accepted 27July 2006

Available online 6September 2006

Abstract

The electrochemical behavior of norethisterone at the mercury electrode was studied in the universal buffer of various pH values using dc-polarography,cyclic voltammetry and controlled-potential electrolysis.Norethisterone was reduced at the mercury electrode via the consumption of two electrons corresponding to reduction of the 3-keto-delta-4-group in the A-ring of the molecule.The p K a value (8.7)of norethisterone was determined from the polarographic and spectrophotometric measurements.A fully validated,simple,sensitive,precise and inexpensive square-wave adsorptive cathodic stripping (SW AdCS)voltammetry procedure was described for trace quanti?cation of bulk norethisterone.The stripping voltammetry peak current of norethisterone in a universal buffer of pH 5following its accumulation onto the hanging mercury drop electrode (HMDE)at ?0.6V (versus Ag/AgCl/KCl s )for 130s showed a linear response with the concentration over the range 5×10?9to 3×10?7M norethisterone.Detection and quantitation limits of 1.5×10?9and 5×10?9M bulk norethisterone,respectively,were achieved.The proposed procedure was successfully applied for the assay of norethisterone in Steronate tablets without interference from excipients.?2006Elsevier B.V .All rights reserved.

Keywords:Norethisterone;Polarography;Cyclic voltammetry;Square-wave adsorptive stripping voltammetry;Quanti?cation;Steronate tablets

1.Introduction

Norethisterone (17-alpha-ethinyl-17beta-hydroxy-4-estren-3-on)is a synthetic progestagenic compound widely used in gynecological practice for contraception and hormone replace-ment therapy in postmenopausal women.It acts to inhibit the secretion of pituitary gonadotrophins that prevents follicular maturation and ovulation [1]

.

(Structure of norethisterone molecule)

Various analytical methods have been described for assay of norethisterone in bulk form,pharmaceuticals and biolog-ical ?uids.These mainly include:high-performance liquid

?

Corresponding author.Tel.:+20106632694;fax:+20403350804.E-mail address:mmghoneim@https://www.360docs.net/doc/1316770794.html, (M.M.Ghoneim).

chromatography [2–7],capillary gas chromatography [8–14],radioimmunological and chromato-mass fragmentography [1],argentometry [15]and differential-pulse polarography [16–19].Adsorptive cathodic stripping voltammetry has been shown as an ef?cient analytical technique for trace determination of a wide range of drugs,which have an interfacial adsorptive char-acter onto surface of the working electrode.However,to date no stripping voltammetric procedure is reported in literature for quanti?cation of norethisterone.

The aim of this study was to clarify the electrochemical behavior of norethisterone at the mercury electrode and to describe a simple and sensitive square-wave adsorptive cathodic stripping (SW AdCS)voltammetry procedure for its quanti?-cation in bulk form and in the pharmaceutical formulation (Steronate tablets).2.Experimental 2.1.Instrumentation

A polarograph (Model 4001Sargent-Welch)was used for the polarographic measurements.A polarographic cell with a drop-

0731-7085/$–see front matter ?2006Elsevier B.V .All rights reserved.doi:10.1016/j.jpba.2006.07.049

500M.M.Ghoneim et al./Journal of Pharmaceutical and Biomedical Analysis43(2007)499–505

ping mercury electrode as a working electrode(m=1.05mg s?1, t=3.3s at mercury height=60cm)and a saturated calomel elec-trode(SCE)as a reference electrode was used.A computer-controlled Electrochemical Analyzers Models263A and394-PAR(Princeton Applied Research,Oak Ridge,TN,USA,) with the software package270/250-PAR were used for the voltammetric measurements.An electrode assembly(303A-PAR)incorporated with a micro-electrolysis cell and a three-electrode con?guration system comprising of a hanging mer-cury drop electrode(HMDE)as a working electrode(surface area=0.026cm2),an Ag/AgCl/KCl s reference electrode and a platinum wire auxiliary electrode was used.A magnetic stirrer (305-PAR)and a stirring bar were used to provide the convec-tive transport of the analyte during its accumulation onto the mercury electrode surface.

A potentiostat/galvanostat(Model173-PAR)incorporated with a digital coulometer Model179-PAR was used for controlled-potential electrolysis.A micro-coulometric cell incorporated with a mercury pool as a working electrode,a sat-urated calomel electrode as a reference electrode and a platinum gauze as a counter electrode was used.The potential applied during the potential-controlled electrolysis was adjusted to be equal to the E1/2of the polarographic wave of norethisterone at the studied pH plus?0.1V.The total charge(Q)passed dur-ing the exhaustive electrolysis of norethisterone was obtained by integrating the current https://www.360docs.net/doc/1316770794.html,ing Faraday’s equa-tion:N=Q/nF(where N is the number of moles of substance being electrolyzed),the number of electrons(n)transferred per norethisterone molecule was determined and was found to be equal to two,which attributed to reduction of the3-keto-delta-4-group in the A-ring of the analyte molecule.

A Shimadzu UV–vis recording Spectrophotometer(Model 160A)was used for the spectral study of norethisterone in buffered solutions.

2.2.Materials and solutions

2.2.1.Norethisterone solutions

Authentic norethisterone was kindly supplied from Schering AG(Berlin,Germany).A stock standard solution of1×10?3M bulk norethisterone was prepared in ethanol(Merck),and then stored at4?C.Working solutions of norethisterone(10?6to 10?4M)were prepared daily by appropriate dilution with ethanol just before use.

2.2.2.Tablet solutions

Twenty tablets of Steronate(Norethisterone acetate;NETA, labeled as containing5mg NETA per tablet—HI Pharm.,Cairo, Egypt)were weighed,and then the average mass per tablet was determined.The tablets were grounded to a homogeneous?ne powder.A quantity of the homogeneous powder of NETA equiv-alent to the weight of two tablets was accurately transferred into a 100ml volume calibrated?ask contains70ml ethanol(Merck). The content of the?ask was sonicated for about10min and then made up to the volume with ethanol.The solution was then?ltered through a0.45?m milli-pore?lter(Gelman,Ger-many).The desired concentrations of the NETA were obtained by accurate dilution with ethanol.The solution was directly ana-lyzed according to the general analytical procedure.A Mettler balance(Toledo-AB104,Switzerland)was used for weighing the solid materials.

2.2.

3.Supporting electrolyte

A series of the universal buffer of pH2–11.5as a supporting electrolyte was prepared[20].A pH-meter(Crison,Barcelona, Spain)was used for the pH measurements.All the chemicals used(Merk)were of analytical-reagent grade and were used without further puri?cation.The de-ionized water was supplied from a Purite-Still Plus de-ionizer connected to an AquaMatic double-distillation water system(Hamilton Laboratory Glass Ltd.,Kent,UK).

2.2.4.General analytical procedure

A known volume of the standard stock solution of norethis-terone was pipetted into a10ml volume calibrated?ask and then made up to the volume with the universal buffer of pH5. The solution was introduced into the electrolysis cell,and then deoxygenated with pure nitrogen gas for about10min in the ?rst cycle and for30s in each successive cycle,while the nitro-gen gas was kept over the solution during the measurements. Accumulation of norethisterone onto the surface of HMDE was performed at?0.7V(versus Ag/AgCl/KCl s)for130s while stirring the solution at400rpm.After an equilibrium time of 5s allowed for the solution to become quiescent,the voltam-mograms were recorded by scanning the potential towards the negative direction using the square-wave waveform.A calibra-tion graph was constructed under the optimized conditions of the procedure.

3.Results and discussion

3.1.Dc-polarography

The dc-polarograms of1.25×10?4M bulk norethisterone in the universal buffer of pH<7.7showed a single2-electron irreversible cathodic wave which may be attributed to reduction of the3-keto-delta-4-group in the A-ring of the norethisterone molecule.On the increase of pH of the medium this wave splits into two waves,the height of the second wave increased at the expense of the?rst one until the latter disappeared com-pletely in solutions of the pH values≥9.5(Fig.1)at which the polarogram exhibited a single2-electron wave at more negative potentials.The cumulative i l–pH curve for the single wave and the?rst splitting one(Fig.2,curve a)or that for the second splitting wave and the single wave(Fig.2,curve b)recalled dis-sociation(Z-shaped)or association(S-shaped)curves,respec-tively,which may be attributed to presence of norethisterone in an acid–base equilibrium[21].The two i l–pH curves(Fig.2, curves a and b)intersected at a pH value equals to the p K a of norethisterone(pH=p K a=8.7)which agrees well with the value(p K a=8.5)obtained from a preliminary spectrophotomet-ric study of3×10?5M norethisterone in the universal buffer of various pH values(pH3–10).

M.M.Ghoneim et al./Journal of Pharmaceutical and Biomedical Analysis 43(2007)499–505

501

Fig.1.Dc-polarograms for 1.25×10?4M bulk norethisterone in the universal buffer of various pH values:(a)2.3;(b)2.7;(c)3.3;(d)4.3;(e)5.3;(f)5.9;(g)6.7;(h)7.7;(i)8.5;(j)8.8;(k)9.2;(l)10.0;(m)10.5;and (n)pH 11.4.

The half-wave potentials (E 1/2),of the single wave recorded in solutions of pH <7.7,and that of the ?rst splitting wave recorded in solutions of pH values 7.7–9.2(Fig.1),shifted toward more negative values with the increase of pH.This behavior indicated the involvement of protons in the rate-determining step and that the proton transfer precedes the electron transfer reaction [21].On the other hand,the E 1/2of both the second splitting wave (pH 7.7–9.2)and the single wave (pH ≥9.5)were practically pH-independent.The linear E 1/2–pH plot for both the single wave at pH values <7.7and the second splitting one at pH values 7.7–9.2and that of the single wave at pH values ≥9.5)intersected at a pH value equals to the p K a value of norethisterone (pH =p K a =8.7)which agrees well with that value obtained from either the i l –pH or the absorbance—pH curves.

Thus,sequence of the electrode reaction of the 3-keto-delta-4-group in the A-ring of norethisterone molecule at the mercury electrode can be expressed as follows [21]:

(i)In acidic and neutral solutions (pH <7.7)the sequence is proton,electron,electron,proton;(ii)in alkaline

solutions

Fig.2.i l –pH plots for 1.25×10?4M bulk norethisterone.(a)The single wave (pH <7.7)and the ?rst splitting wave (pH 7.7–9.2);(b)the second splitting wave (pH 7.7–9.2)and the single wave (pH ≥9.5);(c)total limiting

current.

Fig. 3.Plots of log[(i l ?i HA )/i HA ]and log[(i l ?i ?A )/i ?

A ]vs.pH for 1.25×10?4M bulk norethisterone:(a)?rst splitting wave;(b)second splitting wave recorded in solutions of pH 7.7–9.2.

of pH values ≥9.5the sequence is electron,electron,proton,proton;and (iii)in solutions of intermediate pH values 7.7–9.2norethisterone molecule is expected to present in an acid–base

equilibrium (p K a ≈8.7),AH k d k r A ?+H + K =

k d k r

both the acidic (AH)and basic (A ?)forms are electro-active.Thus,the reduction of norethisterone over the pH range 7.7–9.2takes place via two steps,and the limiting current i l of the sec-ond step (reduction of A ?form)increased at the expense of that of the ?rst one (reduction of AH form)on the increase of pH until the latter one disappeared completely in solutions of pH ≥9.5(Figs.1and 2).Thus,the electrode reaction of norethis-terone over the pH range (7.7–9.2)was considered to consist of mixed processes {(i)and (ii)}.According to Zuman [21],the existence of an electroactive compound in an acid–base equi-librium leads to the variation of the reduction current with the change of hydrogen ion concentration [H +]as governed by the equation:

i AH i l =0.886{(k r t 1/K )[H +]}

1/2

1+0.886{(k r t 1/K )[H +]}

1/2which was modi?ed to the following forms [21,22]:

pH =log 0.886

k r t 1K

1/2

+log i l ?i AH i AH pH =log 0.886

k r t 1

K

1/2

+log

i l ?i ?A

i ?A

where i AH and i ?A are the reduction currents of the acidic and basic forms,respectively.Plots of [log(i l ?i AH )/i AH ]or

[log(i l ?i ?A )/i ?

A ]versus pH,respectively,for the ?rst splitting or the second splitting waves were straight lines (Fig.3)which

502M.M.Ghoneim et al./Journal of Pharmaceutical and Biomedical Analysis 43(2007)499–505

indicated the validity of the previous equations and consequently con?rmed the presence of norethisterone in an acid–base equi-librium within the pH range 7.7–9.2.The pH value corresponds to the intersection point of the two curves (Fig.3,curves a and

b)equals to the p K a of norethisterone (pH =p K a ≈8.7).3.2.Cyclic voltammetry

Cyclic voltammograms of 1.25×10?4M norethisterone at

the HMDE in the universal buffer of pH values 2–5exhibit a single 2-electron irreversible cathodic peak,which attributed to the reduction of the 3-keto-delta-4-group in the A-ring of the acidic form of norethisterone molecule.As the pH of the medium increased,the peak current intensity decreased and a new irre-versible cathodic peak was observed at a more negative potential.Its peak current increased at the expense of the ?rst one until the latter disappeared completely in solutions of the pH values ≥9.5.The single 2-electron cathodic peak observed at the pH values ≥9.5may be attributed to the reduction of the 3-keto-delta-4-group in the A-ring of the basic form of norethisterone molecule.The interfacial adsorptive character of norethisterone was identi?ed by recording the cyclic voltammograms of 1×10?6M in a universal buffer of pH 5following accumulation of the drug onto the HMDE at open circuit (Fig.4,curve a)and at ?0.6V for 60s (Fig.4,curve b)at scan rate 100mV s ?1.The sub-stantial enhancement of the peak current intensity of the ?rst scan (curve b)compared to that of the subsequent scan at the same mercury drop (curve c)or with that recorded following accumulation of the drug at open circuit (curve a)con?rmed the interfacial adsorptive character of norethisterone onto the mercury electrode.The adsorptive character of norethisterone was also identi?ed by measuring the peak current (i p )at vari-ous scan rates ν(50?500mV s ?1)following accumulation

onto

Fig.4.Cyclic voltammograms for 1×10?6M bulk norethisterone in a univer-sal buffer (pH 5)at a scan rate =100mV s ?1,following accumulation onto the HMDE:(a)at open circuit;(b)at E acc.=?0.6V for 60s;and then the repetitive cycle at the same mercury drop (c).

the HMDE for 60s at ?0.6V .A linear log i p versus log νplot following the equation:log i p =0.98,log ν–1.86,r =0.999was obtained.The slope value of 0.98is close to the expected theo-retical value 1.0for an ideal reaction of surface species [23].The electrode surface coverage Γ0(amount of reactant adsorbed onto the mercury electrode surface,mol cm ?2)was cal-culated using the equation:Γ0=Q /nFA ,where Q is the amount of charge consumed by the surface process as calculated by the integration of the area under the peak of the cyclic voltammo-gram of analyte corrected to residual current [24],n is the total number of electrons consumed in the reduction process (n =2)and A is the mercury electrode surface area (0.026cm 2).On dividing the number of coulombs transferred (1102?C)by the conversion factor nFA (5017.3×109?C),a surface coverage of 2.1964×10?10mol cm ?2was obtained.Thus,each adsorbed norethisterone molecule occupies an area of 0.7559nm 2.3.3.Square-wave stripping voltammetry

3.3.1.Optimization of an analytical procedure

The in?uence of pH on square-wave adsorptive cathodic stripping (SW AdCS)voltammetric response for 5×10?7M norethisterone was examined in the universal buffer of var-ious pH values following accumulation onto the HMDE for 60s at E acc.=?0.7V .The voltammograms exhibited a single irreversible 2-electron cathodic peak,its intensity decreased markedly on the increase of pH of the medium.A well-de?ned peak current was achieved at pH 5.Therefore,a universal buffer of pH 5was used as a supporting electrolyte in the rest of the present analytical studies.

The effect of instrumental conditions, e.g.frequency f (10–100Hz),scan increment E s (2–10mV)and pulse-amplitude a (10–50mV)on the peak current intensity of 5×10?7M norethisterone in the universal buffer (pH 5)fol-lowing accumulation onto the HMDE for 60s at E acc.=?0.7V was studied.The achieved optimal instrumental conditions were f =70Hz, E s =10mV and,a =25mV .

The effect of accumulation potential (E acc.)on SW AdCS voltammetry peak current intensity of 5×10?7M norethis-terone was examined over the range ?0.3to ?0.9V following accumulation onto the HMDE for 60s.The peak current inten-sity was practically independent of the accumulation potential up to ?0.6V then decreased.Also effect of accumulation duration (t acc.)at ?0.6V on the peak current intensity of various concen-trations of bulk norethisterone (5×10?7,3×10?7,1×10?7,5×10?8and 3×10?8M)in the universal buffer of pH 5was evaluated.As shown in Fig.5,the response was linear up to 90s for 5×10?7M norethisterone solution and to 130s for 3×10?7and 1×10?7M solutions,then leveled off (curves a,b and c).While for 5×10?8and 3×10?8M norethisterone solutions,as accumulation duration increases,linearity is prevailed over the tested accumulation durations (curves d and e).This means that the lower the concentration of the analyte,the longer of the accumulation duration is.Accordingly,the optimal accu-mulation conditions were E acc.=?0.6V ,and t acc.≤130s.The in?uence of the rest time was also considered and a time period of 5s was chosen.

M.M.Ghoneim et al./Journal of Pharmaceutical and Biomedical Analysis43(2007)499–505

503

Fig.5.Effect of accumulation duration(t acc.),on the SW AdCS voltammetry peak current intensity for:(a)5×10?7;(b)3×10?7;(c)1×10?7;(d)5×10?8; and(e)3×10?8M norethisterone in a universal buffer(pH5);E acc.=?0.6V; f=70Hz, E s=10mV and a=25mV.

3.3.2.Procedure validation

Validation of the proposed procedure for assay of bulk norethisterone was examined via evaluation of limit of detection (LOD),limit of quantitation(LOQ),repeatability,reproducibil-ity,precision,selectivity,robustness and intermediate precision [25].As shown in Fig.6,the stripping voltammetry peak current increased with concentration of norethisterone.Linear calibra-tion graphs over various concentration ranges between5×10?9 and8×10?7M bulk norethisterone,at various accumulation duration(40–130s)were obtained.Characteristics of these cal-ibration graphs are reported in Table1.The LOD and LOQ of norethisterone were estimated at different accumulation dura-tions,using the relation k S.D./b[26],where k=3for LOD and 10for LOQ,S.D.is the standard deviation of the blank(or the intercept of the calibration curve)and b is the slope of the cal-ibration graph.The results reported in Table1,indicated the reliability of the proposed SW AdCS voltammetric procedure for the trace assay of bulk norethisterone.

The repeatability,reproducibility,precision and accuracy [25]of analysis using the proposed procedure were identi?ed by performing three replicate measurements for1×10?7and 3×10?7M bulk norethisterone(t acc.=130s and E acc.=?0.6V) over one day(Intra-day assay)and for three days over a period of one week(inter-day assay).Satisfactory mean percentage

recov-Fig.6.SW AdCS voltammograms in a universal buffer of pH5for various con-centrations of bulk norethisterone:the dotted line represents the background (a)3×10?8;(b)5×10?8;(c)7×10?8;(d)1×10?7;(e)2×10?7;and(f) 3×10?7M;E acc.=?0.6V,t acc.=130s,f=70Hz, E s=10mV and a=25mV. eries(%R)and relative standard deviations(R.S.D.%)were achieved(Table2).

The selectivity[25]of the optimized procedure was tested by analysis of a1×10?7M bulk norethisterone and a standard tablet solution containing1×10?7M norethisterone,follow-ing accumulation onto the HMDE for130s in both cases.No signi?cant differences in the recoveries or the relative standard deviations were observed in the absence(100.20±0.84)and presence(101.11±1.32)of excipients.Thus,the proposed pro-cedure can be considered selective.

Evaluating the in?uence of small variations in some of the most important procedural conditions,including pH(5–5.5), accumulation potential E acc.(?0.5to?0.6V)and accumulation duration t acc.(120–130s)on the recovery of the drug,exam-ined the robustness[25]of the procedure.The results showed that none of these variables signi?cantly affect the recovery of norethisterone and provide an indication of the reliability of the proposed procedure for its assay.Thus,the proposed pro-cedure could be considered robust.Moreover,the intermediate precision[25]of the measurements was examined by assay of bulk norethisterone using two electrochemical analyzers mod-els,263A-PAR,Lab.(1)and394-PAR,Lab.(2)under the same

Table1

Characteristics of the calibration curves of SW AdCS voltammetric determination of bulk norethisterone;pH5,E acc.=?0.6V,f=70Hz, E s=10mV and a=25mV t acc.(s)Linearity range(M)Regression equation i p(?A)=bC(?M)+a S B(r)LOD(M)LOQ(M) 402×10?8to8×10?7i p=2.820C+0.063 5.64×10?30.996 6.0×10?9 2.0×10?8 908×10?9to5×10?7i p=5.144C+0.175 3.99×10?30.999 2.3×10?97.7×10?9 1305×10?9to3×10?7i p=6.831C+0.297 3.42×10?30.996 1.5×10?9 5.0×10?9

504M.M.Ghoneim et al./Journal of Pharmaceutical and Biomedical Analysis43(2007)499–505

Table2

Precision and accuracy of assay of norethisterone by the proposed SW AdCS voltammetry procedure(n=3);t acc.=130s

Conc.(taken)(M)×107Conc.(found)(M)×107R(%)S.D.Accuracy bias(%)Precision%R.S.D. Intra-day

1 1.002100.200.0080.20.84

3 3.012100.400.0550.

4 1.84

Inter-day

1 1.014101.40.016 1.40 1.60

3 3.037101.230.069 1.23 2.87

Table3

Assay of Steronate tablets by proposed SW AdCS voltammetry procedure (t acc.=70s,n=4)and a reported HPLC method[4]

Claimed(mg/tablet) 5.0 Recovery by the proposed method(R%±R.S.D.)

(calibration curve method)

100.15±1.42 Recovery by the proposed method(R%±R.S.D.)

(standard addition method)

100.40±1.59 Recovery by the reported HPLC method(R%±R.S.D.)

(calibration curve method)

100.60±1.85 F-value 1.697

t-test0.386

The theoretical values of F-and t-test at95%con?dence limit(for n1=4and n2=4)are9.28and2.776,respectively.

procedural conditions at different elapsed times by two different analysts.The mean percentage recoveries obtained due to Lab.

(1)to Lab.(2)and even day to day were found reproducible, since there is no signi?cant difference between the recovery or relative standard deviation values.

3.3.3.Analysis of Steronate tablets

The proposed SWAdCS voltammetric procedure was suc-cessfully applied to assay of norethisterone in Steronate tablets without the necessity for samples pretreatment or time-consuming extraction steps prior to the analysis.The obtained results using calibration curve and standard addition[27]meth-ods demonstrated the validity of the proposed procedure for assay of norethisterone in Steronate tablets(Table3).The anal-ysis exhibited satisfactory results,which were statistically com-pared with those obtained by a reported HPLC method[4].Since the calculated F-value(Table3)did not exceed the theoretical one,there was no signi?cant difference between the proposed and reported methods with respect to reproducibility[28].Also, no signi?cant difference was noticed between the two meth-ods regarding accuracy and precision as revealed by t-test[28], Table3.

4.Conclusion

The electrochemical behavior of norethisterone at the mercury electrode was studied and discussed.Its p K a was estimated from the polarographic and spectrophotometric measurements.A validated square-wave adsorptive cathodic stripping voltammetry procedure was described for trace quan-ti?cation of norethisterone in its pharmaceutical formulation (Steronate tablets)The procedure is simple,selective,and precise.

Acknowledgements

The authors express their deep thanks to Dr.Michael Harre, Chemical Development,Schering AG,Berlin,Germany for sup-plying the bulk norethisterone sample used in the present study. The authors express their gratitude also to the Alexander von Humboldt Foundation(Bonn,Germany)for donating the Elec-trochemical Analyzer(263A-PAR)and the personal computer used in the present study to M.M.Ghoneim.

References

[1]L.E.Golubovskaia,E.N.Korenchuk,K.K.Pivnitskii,N.A.Kuzovkova,

N.D.Fanchenko,Probl.Endokrinol.31(1985)72–75.

[2]N.F.Swynnerton,J.B.Fischer,J.Liq.Chromatogr.3(1980)1195–

1204.

[3]J.C.Loo,R.Brien,J.Liq.Chromatogr.4(1981)871–877.

[4]J.A.Gluck,E.Shek,J.Chromatogr.Sci.18(1980)631–636.

[5]R.Gonzalo-Lumbreras,R.Izquierdo-Hornillos,J.Chromatogr.B:Biomed.

Sci.Appl.742(2000)1–11.

[6]G.M.Sundaresan,T.J.Goehl,V.K.Prasad,J.Pharm.Sci.70(1981)

702–704.

[7]Z.Wu, C.Zhang, C.Yang,X.Zhang, E.Wu,Analyst125(2000)

2201–2205.

[8]B.L.Sahlberg,https://www.360docs.net/doc/1316770794.html,ndgren,M.Axelson,J.Steroid Biochem.26(1987)

609–617.

[9]F.Z.Stanczy,S.Roy,Contraception42(1990)67–96.

[10]W.E.Braselton,T.J.Lin,J.O.Ellegood,https://www.360docs.net/doc/1316770794.html,ls,V.B.Mahesh,Am.J.

Obstet.Gynecol.133(1979)154–160.

[11]F.Pommier,A.Siou?,J.Godbillon,J.Chromatogr.B:Biomed.Appl.674

(1995)155–165.

[12]F.Pommier, A.Siou?,J.Godbillon,J.Chromatogr.A750(1996)

75–81.

[13]W.E.Braselton,T.J.Lin,https://www.360docs.net/doc/1316770794.html,ls,J.O.Ellegood,V.B.Mahesh,J.Steroid

Biochem.1(1977)9–18.

[14]M.S.Rizk,N.A.Zakhari,M.I.Walash,S.S.Toubar,C.J.Brooks,R.Ander-

son,Acta Pharm.Nord.(1991)205–210.

[15]F.I.Khattab,F.M.Ashour,M.M.Amer,J.Pharm.Belg.38(1983)147–

155.

[16]L.N.Opheim,Anal.Chim.Acta89(1977)225–229.

[17]L.G.Chatten,R.N.Yadav,S.Binnington,R.E.Moskalyk,Analyst102

(1977)323–327.

[18]D.Cantin,J.Alary,A.Coeur,J.Pharm.Belg.32(1977)255–263.

[19]R.N.Yadav,F.W.Teare,J.Pharm.Sci.67(1978)436–438.

[20]H.T.S.Britton,Hydrogen Ions,fourth ed.,Chapman and Hall,1952,

pp.113–114.

[21]P.Zuman,The Elucidation of Organic Electrode Processes,Academic

Press,New York,1969,pp.20–51.

M.M.Ghoneim et al./Journal of Pharmaceutical and Biomedical Analysis43(2007)499–505505

[22]T.M.Salem,R.M.Issa,A.M.Hindawey,Ann.Chim.(Rome)64(1974)

735–746.

[23]https://www.360docs.net/doc/1316770794.html,viron,L.Roullier,C.Degrand,J.Electroanal.Chem.112(1980)

11–23.

[24]A.Webber,J.Osteryoung,Anal.Chem.Acta157(1984)17–29.

[25]The USA Pharmacopoeia,The National Formulary,Convention Inc.,USP

26,2003,p.2446.[26]https://www.360docs.net/doc/1316770794.html,ler,https://www.360docs.net/doc/1316770794.html,ler,Statistics for Analytical Chemistry,fourth ed.,Ellis-

Howood,New York,1984,p.115.

[27]G.W.Ewing,Instrumental Methods of Chemical Analysis,?fth ed.,

Lippincott-Raven,Philadelphia,1995,p.464.

[28]G.D.Christian,Analytical Chemistry,?fth ed.,John Wiley&Sons Inc.,

USA,1994,p.36.

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.360docs.net/doc/1316770794.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

免疫学的临床应用

免疫学的临床应用有两个方面:一是应用免疫理论来阐明许多疾病的发病机制和发展规律;二是应用免疫学原理和技术来诊断和防治疾病。本章内容主要是后者。此外,免疫学不仅应用于传统的传染病中,而且在肿瘤、自身免疫病、免疫缺陷病、器官移植、生殖免疫等中均广泛应用。 免疫学防治是指应用免疫制剂或免疫调节药物调整机体的免疫功能,对疾病进行预防和治疗。特异性免疫的获得方式有自然免疫和人工免疫两种。自然免疫主要指机体感染病原体后建立的特异性免疫,也包括胎儿或新生儿经胎盘或乳汁从母体获得抗体而产生的免疫。人工免疫则是人为地使机体获得免疫,是免疫预防的重要手段,包括人工自动免疫、人工被动免疫和过继免疫。 人工自动免疫是给机体接种疫苗或类毒素等抗原物质,刺激机体产生特异性免疫。国内常将用细菌制作的人工主动免疫的生物制品称为菌苗,而将用病毒、立克次体螺旋体等制成的生物制品称为疫苗,而国际上把细菌性制剂,病毒性制剂及类毒素统称为疫苗。经人工自动免疫产生的免疫力出现较慢,但免疫力较持久,故临床上多用于预防。人工自动免疫制剂其主要有灭活疫苗、减毒活疫苗、类毒素、以及各种新型疫苗。 人工被动免疫是给机体输入抗体等制剂,使机体获得特异性免疫力,输入抗体后立即获得免疫力,但维持时间短,约2~3周,临床上用于治疗或紧急预防。人工被动免疫的生物制品主要有抗毒素、抗菌血清与抗病毒血清、胎盘球蛋白和血浆丙种球蛋白。 过继免疫治疗是指给患者转输具有在体内继续扩增效应细胞的一种疗法。如给免疫缺陷病患者转输骨髓细胞;给肿瘤患者输入体外激活扩增的特异肿瘤浸润淋巴细胞或非特异性的LAK细胞等。应用时应考虑供者与受者之间HLA型别是否相同,否则输注的细胞会被迅速清除,或者发生移植物抗宿主反应。再如造血干细胞移植:取患者自身或异体骨髓或脐血输入患者,移植物中的多能干细胞可在体内定居、增殖、分化、使患者恢复造血功能和形成免疫力。造血干细胞移植可用于治疗再生障碍性贫血、白血病以及某些免疫缺陷病和自身免疫病等。 在医学制剂影响免疫功能的制剂主要有两类:免疫增强剂和免疫仰制剂。免疫增强剂是指通过不同方式,达到增强机体免疫力的一类免疫治疗药物。临床上常用于治疗与免疫功能低下有关的疾病及免疫缺陷病。免疫增强剂种类很多,按其作用的先决条件可分为三类:一是免疫替代剂,用来代替某些具有免疫增强作用的生物因子的药物。按其作用机制可分为提高巨噬细胞吞噬功能的药物,提高细胞免疫功能的药物,提高体液免疫功能的药物等;按其作用性质又可分为特异性免疫增强剂和非特异性免疫增强剂;按其来源则可分为细菌性免疫增强剂及非细菌性免疫增强剂。二是免疫恢复剂,能增强被抑制的免疫功能,但对正常免疫功能作用不大。常用的免疫增强剂如:卡介苗、短小棒状杆菌、内毒素、免疫核糖核酸、胸腺素、转移因子、双链聚核苷酸、佐剂等。免疫抑制剂是对机体的免疫反应具有抑制作用的药物。能抑制与免疫反应有关细胞的增殖和功能,能降低抗体免疫反应的制剂。常用的免疫抑制剂主要有五类:(1)糖皮质激素类,如可的松和强的松、泼尼松龙等;(2)微生物代谢产物,如环孢菌素和藤霉素等;(3)抗代谢物,如硫唑嘌呤和6-巯基嘌呤等;(4)多克隆和单克隆抗淋巴细胞抗体,如抗淋巴细胞球蛋白和OKT3等;(5)烷化剂类,如环磷酰胺等。 免疫学诊断是指应用免疫学原理和方法对传染病、免疫性疾病等进行和免疫功能进行测定。由于免疫学检测具有高特异性和敏感性,因此常用临床诊断的一种重要手段。目前常用的免疫诊断方法具有体液免疫试验。细胞免疫试验和皮肤试验三种。 抗原抗体反应在体内表现为溶细胞、杀菌、促进吞噬、中和毒素或引起免疫病理损伤等;在体外可出现凝集、沉淀、细胞溶解和补体结合等可见反应。由于抗体主要存在于血清中,临床上多用血清标本进行试验,故体外的抗原抗体反应曾被称为血清学反应。但随着免疫学

三种定量蛋白质组方法比较

A comparative study on the three quantitative methods frequently used in proteomics, 2D DIGE (difference gel electrophoresis), cICAT (cleavable isotope-coded affinity tags) and iTRAQ (isobaric tags for relative and absolute quantification), was carried out. DIGE and cICAT are familiar techniques used in gel- and LC-based quantitative proteomics, respectively. iTRAQ is a new LC-based technique which is gradually gaining in popularity. A systematic comparison among these quantitative methods has not been reported. In this study, we conducted well-designed comparisons using a six-protein mixture, a reconstituted protein mixture (BSA spiked into human plasma devoid of(缺乏)six abundant proteins), and complex HCT-116 cell lysates as the samples. All three techniques yielded quantitative results with reasonable accuracy when the six-protein or the reconstituted protein mixture was used. In DIGE, accurate quantification was sometimes compromised due to comigration or partial comigration of proteins. The iTRAQ method is more susceptible to errors in precursor ion isolation, which could be manifested with increasing sample complexity. The quantification sensitivity of each method was estimated by the number of peptides detected for each protein. In this regard, the global-tagging iTRAQ technique was more sensitive than the cysteine-specific cICAT method, which in turn was as sensitive as, if not more sensitive than, the DIGE technique. Protein profiling on HCT-116 and HCT-116 p53 ?/? cell lysates displayed limited overlapping among proteins identified by the three methods, suggesting the complementary nature of these methods. Keywords: protein quantification ? DIGE ? cICAT ? iTRAQ ? 2D gel ? LC-MALDI TOF/TOF DIGE:荧光差异凝胶电泳 CICAT:同位素亲和标记 iTRAQ:同位素标记相对和绝对定量

常用免疫学检验技术的基本原理

常用免疫学检验技术的基本原理 免疫学检测即是根据抗原、抗体反应的原理,利用已知的抗原检测未知的抗体或利用已知的抗体检测未知的抗原。由于外源性和内源性抗原均可通过不同的抗原递呈途径诱导生物机体的免疫应答,在生物体内产生特异性和非特异性T 细胞的克隆扩增,并分泌特异性的免疫球蛋白(抗体)。由于抗体-抗原的结合具有特异性和专一性的特点,这种检测可以定性、定位和定量地检测某一特异的蛋白(抗原或抗体)。免疫学检测技术的用途非常广泛,它们可用于各种疾病的诊断、疗效评价及发病机制的研究。 最初的免疫检测方法是将抗原或抗体的一方或双方在某种介质中进行扩散,通过观察抗原-抗体相遇时产生的沉淀反应,检测抗原或抗体,最终达到诊断的目的。这种扩散可以是蛋白的自然扩散,例如环状沉淀试验、单向免疫扩散试验、双向免疫扩散实验。单向免疫扩散试验就是在凝胶中混入抗体,制成含有抗体的凝胶板,而将抗原加入凝胶板预先打好的小孔内,让抗原从小孔向四周的凝胶自然扩散,当一定浓度的抗原和凝胶中的抗体相遇时便能形成免疫复合物,出现以小孔为中心的圆形沉淀圈,沉淀圈的直径与加入的抗原浓度成正比。 利用蛋白在不同酸碱度下带不同电荷的特性,可以利用人为的电场将抗原、抗体扩散,例如免疫电泳试验和双向免疫电泳。免疫电泳首先将抗原加入凝胶中电泳,将抗原各成分依次分散开。然后沿电泳方向平行挖一直线形槽,于槽内加入含有针对各种抗原的混合抗体,让各抗原成分与相应抗体进行自然扩散,形成沉淀线。然后利用标准的抗原-抗体沉淀线进行抗原蛋白(或抗体)的鉴别。上述的方法都是利用肉眼观察抗原-抗体反应产生的沉淀,因此灵敏度有很大的局限。比浊法引入沉淀检测产生的免疫比浊法就是利用浊度计测量液体中抗原-抗体反应产生的浊度,根据标准曲线来计算抗原(或抗体)的含量。该方法不但大大提高了检测的灵敏度,且可对抗原、抗体进行定量的检测。

免疫学发展简史

免疫学发展简史 分三个时期:①经验免疫学时期(公元前400年~18世纪末); ②免疫学科建立时期(19世纪~1975年);③现代免疫学时期(1975年至今)。 一、经验免疫学时期(公元前400年~18世纪末) (一)天花的危害 天花是一种古老的、世界流行的烈性传染病,死亡率可高达25%~40%,我国民间早有“生了孩子算一半,得了天花才算全”的说法。患天花痊愈后留下永久的疤痕,但可获得终身免疫。 16世纪由于西班牙殖民者侵略,将天花传播到美洲,墨西哥土著人从16世纪初(1518年)的2000~3000万人到16世纪末减少到100万人,阿茨特克帝国消亡。16世纪中期之后向南进发,在美洲中部毁灭了玛雅和印加文明,随后又毁灭了秘鲁。 (二)人痘苗接种 1.人痘苗接种实践: 中医称天花为“痘疮”,据史书记载人痘苗接种预防天花的方法是在公元前约400年由我们中华民族的祖先建立的。Zinsser微生物学(1988):发明于中国2000多年之前。 明庆隆年间(1567~1572);16~17世纪人痘苗接种预防天花已在全国普遍展开。清康熙27年(1688)俄国曾派医生到北京学习种痘技术。并经丝绸之路东传至朝鲜、日本和东南亚国家,西传至欧亚、北非及北美各国。 1700年传入英国/Momtagu夫人在英国积极推广人痘苗接种中起了重要的作用。 1721~1722年天花在英国爆发流行期间,英国皇家学会在国王的特许下,主持进行了用犯人和孤儿做人痘苗接种的试验,均获得了成功,试验者无一人死于天花。在此基础上,1722年给英国威尔士王子的两个女儿(一个9岁,一个11岁)也进行了人痘苗接种,也都获得成功。 2.人痘苗接种意义:有三个方面: ①能有效预防天花。 ②在接种方法、痘苗的制备和保存建立了一整套完整的科学方法,为以后疫苗的发展提供了丰富的经验和借鉴。 清代吴谦所著的《医宗金鉴·幼科种痘心法要旨》(1742年)中介绍了四种接种法:痘衣法-痘浆法-旱苗法-水苗法。并指出这些方法的优劣:“水苗为上,旱苗次之,痘衣多不应验,痘浆太涉残忍。” 对痘苗保存指出:“若遇热则气泄,日久则气薄,触污秽则气不清,藏不洁则气不正,此蓄苗之法。”“须贮新磁瓶内,上以物密覆

A distance-field-based approach in generating cross-sections for 2-D vessel quantification

A distance-field-based approach in generating cross-sections for 2-D vessel quantification Dong-Goo Kang and Jong Beom Ra * Dept. of EECS, Korea Advanced Institute of Science and Technology (KAIST) 373-1, Guseong-dong Yuseong-gu, Daejeon 305-701, Korea ABSTRACT For accurate determination of thickness-profile in vessel quantification, it is important to find appropriate vessel cross-sections. To obtain vessel cross-sections, a centerline-based approach has been widely used, but it has several inherent problems causing improper cross-sections. First, this approach cannot define cross-sections in a unique way. Second, cross-sections are sensitive to the degree of smoothness of a detected vessel centerline. Third, a small variation in a centerline causes a considerable change in the resultant cross-sections and this phenomenon brings about improper cross-sections in the abnormal vessel of asymmetric structure. Finally, wrong cross-sections may be detected due to the intersection with the other cross-sections in a region of high curvature. In this paper, instead of a centerline, we propose and adopt a complementary geodesic distance field. Then, we detect a sequence of equidistant lines by using the proposed distance field. Finally, we determine cross-sections by refining the obtained equidistant lines. Due to the prospective properties of the proposed distance field, we can alleviate all of the conventional problems and obtain the cross-sections more proper for vessel quantification. Through the intensive simulation using various 2-D synthesized images, we prove that the proposed method provides non-intersecting cross-sections which are insensitive to local variation of geometrical shapes in abnormal vessels. Keywords : Vessel quantification, thickness profile, cross-sections, geodesic distance field, complementary geodesic distance field. 1. INTRODUCTION Stenoses and aneurysms are important risk factors in cardiovascular, cerebrovascular, and peripheral vascular diseases. A stenosis is characterized by the thickened vessel wall and narrowed lumen, and an aneurysm is characterized by the size increase of vessel lumen. Therefore, on the contrary to normal vessels, abnormal vessels with stenosis or aneurysm have non-uniform thickness. According to the morphology of a vessel lumen, stenoses can be classified into the concentric and eccentric types (Fig. 1(a)), and aneurysms can be classified into the berry, saccular, dissecting, and fusiform types (Fig. 1(b))1. (b)berry saccular dissecting fusiform (a)concentric eccentric Figure 1. Classifications of (a) stenoses and (b) aneurysms As vascular imaging technology advances, the morphology of abnormal vessels can be interpreted through examining various kinds of 2-D or 3-D medical images such as conventional or digital subtraction angiography (DSA)2, computed * jbra@ee.kaist.ac.kr; phone +82-42-869-3434; fax +82-42-869-8360; www-isl.kaist.ac.kr Medical Imaging 2005: Physiology, Function, and Structure from Medical Images,edited by Amir A. Amini, Armando Manduca, Proceedings of SPIE Vol. 5746(SPIE, Bellingham, WA, 2005) · 1605-7422/05/$15 · doi: 10.1117/12.595143747

免疫学诊断技术的发展及趋势

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