Ultron ES-OVM Column: A Bonded Ovomucoid Protein Column for

Many proteins demonstrate capability for chiralrecognition and are used as the basis for chiralHPLC columns. The protein ovomucoid (molecularwight – 28,000) exhibits especially useful qualitiesfor discriminating between enantiomers. Throughthe reaction with N, N1–disuccinimidyl carbonate,this protein has been immobilized on 5-µm aminopropylsilane-derivatized silica particles with120Å pores to form the packing in Ultron ES-OVMcolumns. This covalently bonded ovomucoid columnis extremely useful for performing a wide variety ofchiral separations with acidic, basic, and neutralcompounds [1—10].

This technical overview describes some of the chro-matographic properties of the Ultron ES-OVMcolumn, and includes practical hints for optimizingthe development of chiral separation methods.Included are illustrative applications that show thecapability of this HPLC column for separating enan-tiomers of many different structures.

Ultron ES-OVM Column: A Bonded Ovomucoid Protein Column for Direct HPLCSeparation of Chiral Compounds

Technical Overview

Effect of Mobile-Phase pH on Retentionand Enantioselectivity

The isoelectric point, pI, of ovomucoid is 3.8 to 4.3.Therefore, Ultron ES-OVM packing assumes negativeor positive charges when the pH of the mobile phaseis above or below the pI of ovomucoid, respectively.The practical result is that the pH of the mobilephase used in the separation significantly affectsthe retention and selectivity of enantiomeric com-pounds. The effect of mobile-phase pH on the capac-ity factor (k'1) of the first enantiomer peak and onenantioselectivity (α) was determined for an acidiccompound, ibuprofen (pKa = 4.4); a neutral com-pound, hexobarbital (neutral in the range pH 3.0 to6.0); and a basic compound, chlorpheniramine (pKa = 4.0, 9.2). Structures of these compounds areshown in Figure 1.

CHCH2 CH COOH

CH3H3C

H3CCHCH2 CH

CH3H3C

H3CCOO

_pKa = 4.4

Ibuprofen

CH3

CH3

pKa = 4.0 C

NHCH2CH2NH

H

++

Cl CH3

CH3

pKa = 9.2 C

NCH2CH2NH

H

+Cl CH3

CH3

CN

CH2CH2N

H

Cl

Chlorpheniramine

CH3

H3C

O_

NH

O N

O

O

CH3

H3C N

O N

O

pKa = 8.2

Hexobarbital

Figure 1. Acid-base equilibria of ibuprofen, chlorpheniramine, and hexobarbital.

2

For a mobile phase of 20 mM phosphate buffer/ethanol (100/5), the k'1 of the first enantiomer peakand the separation factor (α = k'2/k'1) of each testracemic compound for pH 3.0 to 6.0 are shown inFigures 2 and 3, respectively. Ovomucoid has a posi-tive charge at pH 3.0, is neutral at pH 4.0 to 4.6, andis negatively charged at pH 5.5. Figure 2 shows thatretention for uncharged hexobarbital is essentiallyconstant throughout the pH range 3.0 to 6.0. The k'1value of the first-eluted enantiomer for this com-pound is slightly above zero, probably due to a weakhydrophobic interaction with immobilized ovomucoid.

increased hydrophilicity of ovomucoid at this pH;however, hydrophobic interaction still dominatesretention.

Figure 2 further shows that the k'1 of chloropheni-ramine increases at higher pH values. Both chlor-pheniramine and ovomucoid are positively chargedat pH 3.0, so electrostatic repulsion dominates andkeeps retention low. This compound is morestrongly retained at pH values higher than the pI ofovomucoid because of strong electrostatic forcesplus the usual hydrophobic interaction.

Figure 3 shows that the enantioselectivity factors (α)for basic compounds can vary strongly with pH. Theα value for chlorpheniramine increases sharply withpH increase; values for the other two compoundschange only slightly.

These results indicate the importance of establish-ing the mobile phase pH that will produce the opti-mum chiral separation of ionic enantiomericcompounds. The effects just discussed imply thatretention by the ovomucoid stationary phase isdominated by two mechanisms: electrostatic andhydrophobic interactions. Since electrostatic inter-actions are strongly influenced by the ionic strengthof the mobile phase, reliable and reproducible sepa-rations are only obtained by careful control of bothpH and salt concentration in the mobile phase.

60

50

40

30k’

20

10

02 3 4 5

pH

6 7

Enantiomeric samples

Conditions Column: Ultron ES-OVM

Column size: 6.0 × 150 mm

Mobile phase: 20 mM KH2PO

4 (pH 4.6)/C

2H

5OH (100/20 v/v)

Flow rate: 1.0 mL/min

Pressure: 110 kg/cm2

Temperature: 25 °C

Detection: UV-220 nm

Ibuprofen

Chlorpheniramine

Hexobarbital

Figure 2. Change in k’ with change in pH.

For ibuprofen, maximum k'1 values are in the rangepH 4.0 to 4.6, as shown in Figure 2. Since the pKa ofibuprofen is 4.4, the compound is partially chargedwhile ovomucoid is uncharged in this pH range;therefore, retention is likely due to strong hydropho-bic interactions between the solute and the proteinstationary phase. The decrease in k'1 at higher pH(for example, 5.0 to 6.0) probably is the result ofion-repulsion of the negatively charged compoundfrom the negatively charged protein stationaryphase. The larger k'1 values of ibuprofen comparedto hexobarbital in the pH range 5.0 to 6.0 is consid-ered to be due to a somewhat stronger hydrophobicinteraction of ibuprofen. The k'1 value of ibuprofendecreases at pH 3.0, presumably because of




4 (pH 4.6)/C

2H

5OH (100/20 v/v)



Temperature: 25 °C


3

2

α

1

02 3 4 5

pH

6 7

Enantiomeric samplesIbuprofen

Chlorpheniramine

Hexobarbital

Figure 3. Change in αα with change in pH.

3

Effect of Mobile Phase Ionic Strength onRetention

As discussed above, ionic strength partly influencesretention on the ovomucoid stationary phase. Thiseffect is demonstrated in Figure 4 for ketoprofen(pKa = 3.9) and chloropheniramine (pKa = 4.0, 9.2).Here, retention of these compounds is shown forthree concentrations of pH 5.5 phosphate buffer (5, 20, 70 mM)/ethanol (100/10). About the same k' values occur for both compounds with 20 and 70 mM buffer, but larger k' values are shown for thenegatively charged ketoprofen at 5 mM buffer. Thiseffect may arise from the strong electrostatic inter-action that occurs at low ionic strength, as iscommon in ion-exchange chromatography. Resultssuch as those just described, combined with the ten-dency of buffers to precipitate with higher concen-trations of organic mobile-phase modifier, suggestthat 20 mM phosphate buffer my be appropriate formany separations. (Note that potassium salts aregenerally more soluble than sodium salts where solubility must be considered.)

Effect of Organic Modifiers on Retentionand Enantioselectivity

The type of organic modifier in the mobile phasecan have a strong effect on both retention (k') and enantiomer band spacing (α) values for chiral com-pounds with the Ultron ES-OVM column. Chiral sep-arations of tolperison (pKa = 8.9) were performedwith mixtures of phosphate buffer (pH 4.6) and fourdifferent modifiers: methanol, ethanol, isopropanol,and acetonitrile. Figure 5 shows that different sepa-rations were produced with the various modifiers.At the same organic concentration (v/v), the elutionstrength order of acetonitrile > isopropanol >ethanol > methanol was observed.



Mobile phase: 20 mM phosphate buffer (pH 5.5)/ethanol

(100/10 v/v); buffer concentration = 5, 10, 70



Temperature: 25 °C


30

20

k’

10

00 20 40 60

Phosphate buffer (mM)

80

Enantiomeric samplesFirst chlorpheniramine

Second chlorpheniramine

First ketoprofen

Second ketoprofen

Figure 4. Effect of ionic strength of phosphate buffer on retention.



Mobile phase: 20 mM phosphate buffer (pH 4.6)/organic modifier

(100/10 v/v)



Temperature: 25 °C


Time (min) Time (min) Time (min) Time (min)

Acetonitrile Ethanol

Isopropanol Methanol

Figure 5. Effect of type of organic modifiers on elution profileof enantiomeric tolperison.

4

Differences in values could be due to the conforma-tional change in ovomucoid, causing structuralchanges in the recognition site(s) for different com-pounds. In this regard, it was reported that the elu-tion order of propranolol enantiomers was reversedby changing organic modifiers [5].

These and other studies indicate that retentioncharacteristics of chiral compounds can be signifi-cantly influenced by the type and concentration oforganic modifier used in the mobile phase. Differentorganic modifiers are favored for different com-pound structures. The optimum organic modifiershould be determined for each system.

Table 1. Effect of Type and Contents of Organic Modifiers in the Mobile Phase on the Capacity Factors and Enantioselectivities on Ultron ES-OVM Column

Ethanol content Acetonitrile content

Acidica Ibuprofen 24.76 1.45 12.38 1.40 24.29 1.25 3.10 1.06

Basica Chloropheniramine 4.10 2.60 1.60 1.93 3.25 2.00 2.00 1.00

Unchargeda Hexobarbital 1.70 1.52 * 1.15 1.23 *

aMobile phase used was a mixture of 20 mM phosphate buffer (pH 4.6) and ethanol or acetonitrile (v/v).

*Hexobarbital did not retain.

5 10 5 10Type of drug Compound k’1 αα k’1 αα k’1 αα k’1 αα

The data in Table 1 show that changes (for example,5 v/v) in organic modifier concentration can changeretention profiles drastically. These results wereobtained for chiral separations of acidic ibuprofen,neutral hexobarbital, and basic chlorpheniraminewith mobile phase mixtures of 20 mM phosphatebuffer (pH 4.6) and ethanol and acetonitrile in theratio of 100/5 and 100/10 (v/v).

Compared to acetonitrile, ethanol as a modifier pro-duced higher values for these test compounds whenorganic concentrations were used to obtain essen-tially the same k' values. (In Table 2, roughly equiva-lent k' values were obtained by mixing appropriateratios of phosphate buffer and organic modifier.)

Table 2. Change in the Enantioselectivity of Enantiomeric Compounds in Mobile Phases Containing Ethanol or Acetonitrile at Equivalent Solvent Strength

Ethanol Acetonitrile

Acidica Ibuprofen 12.40 1.40 13.20 1.16

Basicb Chloropheniramine 8.00 2.14 7.80 1.46

Unchargedc Hexobarbital 2.19 1.59 1.85 1.42

aMobile phase used was a mixture of 20 mM phosphate buffer (pH 4.6) and ethanol (100/10, v/v) or acetonitrile (100/7, v/v).

bMobile phase used was a mixture of 20 mM phosphate buffer (pH 5.5) and ethanol (100/10, v/v) or acetonitrile (100/8, v/v).

cMobile phase used was a mixture of 20 mM phosphate buffer (pH 6.0) and ethanol (100/5, v/v) or acetonitrile (100/4, v/v).

Type of drug Compound k’1 αα k’1 αα

5

Effect of Column Temperature on Retention and Enantioselectivity

Temperature can also be used to optimize the retention and selectivity characteristics of theUltron ES-OVM column. Table 3 shows the effect oftemperature on the chromatographic characteristicsof propranolol enantiomers separated with a mobilephase of 20 mM phosphate buffer (pH 4.6)/ethanol(100/10, v/v) at various temperatures between 5°and 40 °C. Table 3 summarizes the k' and α values;the plate number values, N; and the resolutionvalues, Rs, found for the propranolol enantiomers inthis study. With temperature increase, k' and αvalues decreased while N values increased, the high-est resolution value occurring at 19 °C. Other stud-ies have shown that certain compounds exhibit bestresolution at higher temperatures, while others arebetter resolved at lower temperatures. Therefore, tooptimize any chiral separation on ES-OVM, theeffect of temperature should be investigated.

of lorazepam on ovomucoid takes place at 15 °C orhigher. Actual enantiomer composition is deter-mined by operating the column at 7 °C [8].

Effect of Sample Loading

As with most HPLC columns, measured platenumber is a function of the amount of sampleinjected on the column. The effect of sample mass isillustrated in Figure 7 for a 4.6 × 150 mm Ultron ES-OVM column. These chromatograms wereobtained by increasing sample concentration at afixed volume (0.5 µL) to increase sample loading.Studies have indicated that sample loadings of 1.5to 3 nmol/g packing produced a decrease of 15% orless in enantiomer resolution (30% decrease incolumn plate number) with the 4.6 × 150 mm UltronES-OVM column [5]. Samples of about 2 nmol (1 µg)are typical for most analytical separations on thiscolumn. These results show that small sample load-ings should be used to obtain the sharpest peaksand highest resolution.

The effect of sample volume on column efficiencyand resolution is similar to that for other HPLCcolumns of equivalent efficiency. Sample volumes inthe 1 to 50 µL range generally are permitted, with10 or 25 µL volumes used in typical applications [4].As in all HPLC separations, sample loading is mostcritical when peaks elute at low k' values. Samplemass and volume are less critical and can usually beincreased when peaks are adjusted to elute athigher k' values.

Table 3. Effect of Column Temperature on the ChromatographicParameters of Propranolola

5 °C 11 °C 19 °C 28 °C 40 °C

k’1 18.67 11.49 7.79 4.97 2.59

k’2 23.94 14.59 9.68 6.05 3.06

α 1.28 1.27 1.25 1.22 1.18

N1 446 722 1059 1224 1899

N2 380 563 798 890 1282

Rs 1.12 1.35 1.44 1.21 1.08

aMobile phase used was a mixture of 20 mM phosphate buffer (pH 4.6) and ethanol

(100/10, v/v).

Precise temperature control of the Ultron ES-OVMcolumn is recommended to ensure good repro-ducibility of separations. This characteristic is illustrated by the data in Figure 6 for the chiral separation of trimipramine maleate. With a mobilephase of pH 4.6 phosphate buffer (20 mM)/acetoni-trile (100/20, v/v), column temperatures in thenarrow range of 18 to 25 °C gave significant differ-ences in retention times, although α values wereessentially constant in this case.

In some instances, racemization of the enantiomerscan occur at higher temperatures, and lower operat-ing temperatures are required for determining trueenantiomer composition. This situation is illustratedin the separation of lorazepam with a mobile phaseof pH 4.6 phospate buffer (20 MM)/isopropanol(100/10, v/v). Peak coalescence due to racemization

18 °C 22 °C 25 °C

t2 = 19.93 t2 = 16.35 t2 = 14.13

t1 = 9.65 t1 = 8.28 t1 = 7.29

Time (min) Time (min) Time (min)



Mobile phase: 20 mM phosphate buffer (pH 4.6)/acetonitrile (100/20 v/v)



Temperature: Varied


Figure 6. Effect of column temperature on the separation ofenantiomeric trimipramine maleate.

6

Effect of Mobile Phase Flow Rate onColumn Efficiency

As is typical of other HPLC columns, column effi-ciency is influenced by the mobile phase flow rateused in the separation. Figure 8 shows plate heightversus flow rate plots for two Ultron ES-OVMcolumns of different internal diameters. These andother results suggest that a flow rate of about 1.0 mL/min often produces the highest column effi-ciency and peak resolution for these two columns.The flow rate for the highest column efficiency actu-ally is dependent on the type and concentration oforganic modifier and the temperature of operation.For best results, the optimum flow rate should bedetermined for each separation system.

10 10 10

Time (min)

0.75 µg 1.5 µg 5 µg 10 µg 15 µg

10 1020

Sample loading amount




4 (pH 5.5)/C

2H

5OH (100/4 v/v)


Temperature: Ambient


Injection volume: 5-uL loop

Sample: Tolperison

Figure 7. Effect of sample loading – tolperison.

0.16

0.14

0.12

0.10

0.08

0.06

0.04

0.02

0.50 0.75 1.00 1.25

Mobile phase flow rate

Pla

te h

eig

ht

cm

1.50 1.75 2.00

4.6 mm id

6.0 mm id

Figure 8. Influence of flow rate on plate height, Ultron ES-OVM

columns, 150 mm

7

Optimizing Chromatographic Conditionsfor Ultron ES-OVM Column

Suggested steps for optimizing a separation of enan-tiomers are given in Figure 9. An initial temperatureof 25 °C is recommended, since higher tempera-tures could racemize the chiral compounds,decrease resolution, and may also lead to denatura-tion of the ovomucoid stationary phase. An initialmobile phase of 20 mM phosphate with acetonitrileorganic modifier is suggested [5]. For acidic enan-tiomeric compounds, a starting pH of 3.0 is recom-mended – α values generally are constant in the pHrange 3.0 to 4.6, and faster separations usually areavailable at low pH. If the desired separation is not

attained, increase the pH to 4.0, then, if needed, addadditional organic modifier. If the desired separa-tion still is not obtained, follow the same steps witha different modifier or with a different buffer (otherthan phosphate).

For basic or neutral enantiomers, an initial mobilephase of phosphate buffer pH 4.6/acetonitrile is rec-ommended. If the desired separation is not obtained,raise the pH to 7.0 and increase the concentration oforganic modifier to improve the separation. If thedesired separation still is not obtained, follow thesame steps with a different organic modifier or witha different buffer (other than phosphate) asdescribed above for acidic samples.

Acidic compounds

Begin with 0.02 M phosphate buffer (pH 3.0)

Add uncharged modifier (max 50%)*Increase pH to 4.0

(No or low enantioselectivity)

Increase pH to 4.0 and/or try another

uncharged modifier

Try another buffer*

(Separation)

(No peak eluted)(No or low enantioselectivity)

Basic and nonprotolytic compounds

(Separation)

Try another buffer*

(No or low enantioselectivity)(Separation)

Begin with 0.02 M phosphate buffer (pH 4.6)

Add uncharged modifier (max 50%)*Increase pH to 4.6 to 7.0

(No or low enantioselectivity)

Increase pH to 4.6 to 7.0 and/or try

another uncharged modifier

Try another buffer*

(Separation)

(No peak eluted)(No or low enantioselectivity)

(Separation)

Try another buffer*

(No or low enantioselectivity)(Separation)

1)

2)

1)2)

1)

2)

1)2)

*Uncharged modifier: Acetonitrile, ethanol, methanol, 2-propanol

Buffer solution (20 mM): KH2PO4/K2HPO4, HCOOH/HCOONH4, CH3COOH/CH3COONa, C3H4OH(COOH)3KH2PO4

8

Table 4 summarizes the resolution and optimizedmobile phase that were obtained for 43 enan-tiomeric pairs, using the approach of Figure 9.Other useful optimization approaches for the UltronES-OVM column are given in References 4 and 5.

Substance Mobile Phase Rs

Acetylpheneturide 20 mM phosphate buffer (pH 4.6)-ethanol (100/7.5 v/v) 2.74

Alimemazine 20 mM phosphate buffer (pH 4.6)-ethanol (100/25 v/v) 6.06

Bay K 8644 20 mM phosphate buffer (pH 4.6)-ethanol (100/25 v/v) 5.92

Benproperine 20 mM phosphate buffer (pH 4.6)-ethanol (100/20 v/v) 3.27

Benzoin 20 mM phosphate buffer (pH 4.6)-ethanol (100/10 v/v) 8.41

Biperiden 20 mM phosphate buffer (pH 4.6)-ethanol (100/10 v/v) 3.17

Bunitrolol 20 mM phosphate buffer (pH 6.0)-ethanol (100/3 v/v) 3.08

Bupivacaine 20 mM phosphate buffer (pH 5.5)-acetonitrile (100/10 v/v) 1.26

Chlormezanone 20 mM phosphate buffer (pH 4.6)-ethanol (100/10 v/v) 6.48

Chlorphenesin 20 mM phosphate buffer (pH 5.5) 2.23

Chlorpheniramin 20 mM phosphate buffer (pH 5.0)-acetonitrile (100/5 v/v) 2.36

Chlorprenaline 20 mM phosphate buffer (pH 5.5)-ethanol (100/3 v/v) 2.34

Cloperastin 20 mM phosphate buffer (pH 4.6)-ethanol (100/15 v/v) 2.85

Dimetindene 20 mM phosphate buffer (pH 4.6)-ethanol (100/15 v/v) 4.33

1,2-Diphenylethylamine 20 mM phosphate buffer (pH 5.5) 1.74

Disopyramid 20 mM phosphate buffer (pH 5.5)-ethanol (100/10 v/v) 2.04

Eperisone 20 mM phosphate buffer (pH 5.5)-ethanol (100/5 v/v) 1.15

Ethiazide 20 mM phosphate buffer (pH 4.6) 1.42

Flurbiprofen 20 mM phosphate buffer (pH 3.0)-ethanol (100/30 v/v) 1.26

Glutethimide 20 mM phosphate buffer (pH 4.6)-ethanol (100/10 v/v) 1.36

Glycopyrronium 20 mM phosphate buffer (pH 4.6) 1.73

Hexobarbital 20 mM phosphate buffer (pH 5.5)-ethanol (100/5 v/v) 1.70

Homochlorcyclizine 20 mM phosphate buffer (pH 4.6)-ethanol (100/10 v/v) 3.04

Hydroxyzine 20 mM phosphate buffer (pH 4.6)-ethanol (100/15 v/v) 2.15

Ibuprofen 20 mM phosphate buffer (pH 3.0)-ethanol (100/10 v/v) 1.73

Ketoprofen 20 mM phosphate buffer (pH 3.0)-acetonitrile (100/10 v/v) 1.37

Meclizine 20 mM phosphate buffer (pH 4.6)-ethanol (100/35 v/v) 3.71

Mepenzolate 20 mM phosphate buffer (pH 4.6) 1.40

Mephobarbital 20 mM phosphate buffer (pH 4.6)-ethanol (100/10 v/v) 1.70

Methylphenidate 20 mM phosphate buffer (pH 5.7) 1.13

Oxprenolol 20 mM phosphate buffer (pH 5.5)-ethanol (100/10 v/v) 1.38

Pindolol 20 mM phosphate buffer (pH 5.5)-ethanol (100/3 v/v) 2.04

Pranoprofen 20 mM phosphate buffer (pH 3.0)-acetonitrile (100/8 v/v) 1.01

Prenylamine 20 mM phosphate buffer (pH 5.0)-acetonitrile (100/15 v/v) 1.02

Profenamine 20 mM phosphate buffer (pH 4.6)-ethanol (100/25 v/v) 3.31

Proglumide 20 mM phosphate buffer (pH 4.6)-ethanol (100/20 v/v) 1.32

Promethazine 20 mM phosphate buffer (pH 4.6)-ethanol (100/20 v/v) 0.98

Propranolol 20 mM phosphate buffer (pH 6.8)-acetonitrile (100/30 v/v) 1.24

Thioridazine 20 mM phosphate buffer (pH 5.5)-acetonitrile (100/30 v/v) 0.98

Tolperizone 20 mM phosphate buffer (pH 5.5)-ethanol (100/10 v/v) 1.50

Trihexyphenidyl 20 mM phosphate buffer (pH 4.6)-ethanol (100/10 v/v) 5.16

Trimipramine 20 mM phosphate buffer (pH 4.6)-ethanol (100/30 v/v) 3.69

Verapamil 20 mM phosphate buffer (pH 4.6)-ethanol (100/5 v/v) 1.49

Table 4. Separation of Enantiomeric Pairs with Ultron ES-OVM

9

Column Care

Buffered mobile phases with pH ranges of 2 to 7.5 (3 to 7 preferred) and common organic water-misci-ble solvents (for example, acetonitrile, methanol,ethanol, and propanol) can be used safely with theUltron ES-OVM column. Mobile phases with > 50%organic solvent should be used sparingly. Guardcolumns are recommended for most applications, toprotect the analytical column against deleteriouscontaminants. Samples containing extraneous mate-rials that will be highly retained on the columnshould be carefully avoided. Contaminated columnsexhibiting poor peak shapes sometimes are conve-niently restored by flushing with 20 to 40 columnvolumes of 50% acetonitrile/distilled water.Columns are preferably stored in a mobile phase of10 to 20% acetonitrile/distilled water (after flushingout any previously used buffer) when not in use forlong periods.

Illustrative Applications

In the appendix are chromatograms of a wide rangeof enantiomeric compounds that have been success-fully separated with the Ultron ES-OVM column. Ineach case, no attempt was made to optimize results,the main criterion being a separation that wouldhave distinct utility in the characterization ofracemic mixtures. Also included is a separation on alarger diameter column (6.0 × 150 mm), which canaccept semipreparative samples (Figure 10).

References1. T. Miwa, T. Miyakawa, and M. Kayano, Chem.

Pharm. Bull., 35 (1987) 682.

2. T. Miwa, T. Miyakawa, and M. Kayano, J. Chro-matogr., 408 (1987) 316.

3. M. Okamoto and H. Nakzawa, J. Chromatogr.,504 (1990) 445.

4. Y. Hu and D. Kupfer, Drug Metabolism and Dis-position, 30 (2002) 1329.

5. K. M. Kirkland, K. L. Neilson, D. A. McCombs,and J. J. DeStefano, LC-GC, 10 (1992) 322.

6. K. Ishii, S. Wakamoto, H. Nakai, and T. Sata,Chromatographia, 43 (1996) 413.

7. K. M. Kirkland, K. L. Neilson, and D. A.McCombs., J. Chromatogr., 545 (1991) 43.

8. Y. Hu and D. Kupfer, Drug Metabolism and Dis-position, 30 (2002) 1329.

9. J. Haginaka, J. Wakai, K. Takahashi, H. Yasuda,and T. Takagi, Chromatographia, 29 (1990)587.

10. P. Bonato, R. Bortocan, C. Gaitani, F. Paias, M.Ina, and R. Lima, J. Braz. Chem. Soc., 13 (2),2002.

For More Information

For more information on our products and services,visit our Web site at www.agilent.com/chem.




4 (pH = 4.6)/C

2H

5OH = 100/20 v/v



Temperature: 25 °C


10 20

Time (min)

S

N

2CH CH

CH3

NCH3

CH3

Figure 10. Promethazine.

10

Appendix

10

Time (min)

20 30

AntiinflammatoryPranoprofen

N O

CH

CH3

COOH*




4 (pH 6.0)/

C2H

5OH (100/5 v/v)


Temperature: 27 °C

Detection: UV-220 nm (0.04 AUFS)




4 (pH = 4.6)


Temperature: 27 °C


10

Time (min)

20 30

Cholinergic blocking(parasympatholytic) drugsMepenzolate bromide

C

OHH C3 CH3

COO * Br_

+N




4 (pH 5.6)/

C2H

5OH (100/10 v/v)


Temperature: 27 °C


10 40

Time (min)

5020 30

AntiinflammatoryFenoprofen calcium

O

CHCH3

COO*

_

Ca3+ 2H2O

10

Time (min)

20

AntihistaminesPromethazine hydrochloride



Mobile phase: 20 mM KH2PO4 (pH 4.6)/

C2H5OH (100/20 v/v)




O

S

CH2

CH3

CH3CH N HCI*

10

Time (min)

20

Local AnestheticBupivacaine




4 (pH 5.5)/

CH3CN (100/10 v/v)


Temperature: 25 °C


N

(CH2)3CH3

CN

O

CH3

CH3

*H

N CH2 CH2 C

OH

HCI*

5

Time (min)

10

Antiparkinson drugsTrihexyphenidyl




4 (pH 4.6)/

C2H

5OH (100/10 v/v)




11

10

Time (min)

20

Skeletal muscle relaxantsEperisone hydrochloride




4 (pH 5.5)/

C2H

5OH (100/5 v/v)


Temperature: 25 °C


H3CH2 C CH CH2 N

O CH3

HCl**CC

NH

NH

H

S

C H5

O

Cl

SNH2

OO O

*

10Time (min)

20

Diuretic drugsEthiazide




4 (pH 4.6)


Temperature: 25 °C


2

10 40

Time (min)

20 30

AntipsychoticsThioridazine hydrochloride




4 (pH 5.5)/

CH3CN (100/30 v/v)


Temperature: 31 °C


S

N SCH3

CH2 CH2

NCH3 HCI*

*

10

Time (min)

20 30

1,2-Diphenylethylamine




4 (pH 5.5)


Temperature: 25 °C


CH2 CH

NH2

*

10

Time (min)

20 30 40

Prenylamine lactate




4 (pH 5.0)/

CH3CN (100/15 v/v)


Temperature: 25 °C


CH (CH2)2 NH CH CH2

CH3

*

2-Phenyl propionic acid




4 (pH 4.6)


Temperature: 25 °C


CH COOHCH3*

10

Time (min)

20

12

Naftopidil (Alpha1a-adrenergic

receptor antagonist)

O CH2CHCH2

OH

N N

H3CO

7.4

04

10

.19

3

2015105

Minute

0

Conditions Column: Ultron ES-OVM (5 µm)



CH3CN (75/25 v/v)


Temperature: 25 °C


Sample: Naftopidil 0.5 mg/mL

Injection vol: 1 µL

8.0

60

15

.20

0

Bicalutamide (Antiandrogen)

F SO2 CH2 C C

OH

N

O

H

CF3

CN

H3C

0 5 10

Minute

15 20 25




CH3CN (88/12 v/v)


Temperature: 25 °C


Sample: Bicalutamide in CH3CN

0.1 mg/mL


302010Minute

0

NN

NO2

CH3

CH3

OP ONaOONa 2

0.2

08

23

.12

8




CH3CN (88/12 v/v)


Temperature: 25 °C


Sample: Ornidazol disodium phosphate

0.2 mg/mL


Ornidazol (Antibiotic)

Cetirizine hydrochloride(Antihistamine)

CH

Cl

N N CH2CH2OCH2COOH 2HCI

10

.70

0

12

.59

5

2015105

Minute

0




CH3CN (90/10 v/v)


Temperature: 25 °C


Sample: Cetirizine hydrochloride

0.2 mg/mL


Fulvastatin sodium(HMG-CoA reductase inhibitor)

34

.54

6

39

.91

3

F

N

OHCOONa

Minute




4 (pH 3.0)/

CH3CN (88/12 v/v)


Temperature: 25 °C


Sample: Fulvastatin sodium 0.5 mg/mL

(Solvent: methanol)


0 10 20 30 40 50 60

Amlodipine besilate(Calcium channel blocker)

HN CH2OCH2CH2NH2

CH3

COOCH3 COOC2H5

Cl

.

SO3H

6.7

83

11.3

31

0 5 10

Minute

15 20



Mobile phase: 10 mM Na2HPO4 (pH 7.0)/

CH3CN (78/22 v/v)


Temperature: 25 °C


Sample: Amlodipine besilate

0.02 mg/mL (Solvent: Methanol)


13

22

.58

7

27.

81

2

Minute

Sodium rabeprazole(Proton pump inhibitor)

N

OH2CH2CH2COH3C CH3

CH2S

N

Na

O

0 10 20 30 40




CH3CN (95/5 v/v)


Temperature: 25 °C


Sample: Sodium rabeprazole

0.2 mg/mL


N

Fexofenadine hydrochloride(Antihistaminic)

COH N (CH2)3 CH

OH

C

CH3

CH3

COOH HCl7

.31

3

8.7

18

0 4 8 12 16




CH3CN (95/5 v/v)


Temperature: 25 °C


Sample: Fexofenadine hydrochloride

0.3 mg/mL


Agilent shall not be liable for errors contained herein or for incidental or consequential

damages in connection with the furnishing, performance, or use of this material.

Information, descriptions, and specifications in this publication are subject to change

without notice.

© Agilent Technologies, Inc. 2008

Printed in the USA

July 11, 2008

5989-8748EN

www.agilent.com/chem

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