1determination of drugs in blood plasma by hplc for...

Asian Journal of Drug Metabolism and Pharmacokinetics Paper ID 1608-2281-(2004)-0403-00177-12 Copyright by Hong Kong Medical Publisher Received October 12, 2003I SSN 1608-2281 2004; 4(3): 177-188 Accepted May 10 , 2004

Determination of drugs in blood plasma by HPLC for biological samples, using on-line sample pretreatment technique Yuki Hashi, Masatoshi Takahashi, Keiko Yayabe, Yoshihiro Hayakawa and Hirohisa Mikami Analytical Applications Department, Analytical and Measuring Instruments Division, Shimadzu Corporation, Kyoto, Japan Abstract We have developed a new high performance liquid chromatograph (HPLC) system, which

utilizes an on-line sample pretreatment technique, for the analyses of drugs in biological samples such as blood plasma and serum. This new system, named the "Co-Sense for BA", is an HPLC system which employs the automated column switching technique, the newly developed Shim-pack MAYI-ODS sample pretreatment column, and the new on-line dilution method. The blood plasma or serum sample, containing drugs, was injected directly into the Shim-pack MAYI-ODS column without pretreatment, and then on-line diluted and on-line deproteinized in a complete automated sequence by the system. After the on-line pretreatment, the drug components were introduced into the HPLC analytical column for separation and determination. This report describes the principle, of the newly developed HPLC system and its application to analyses of drugs in blood plasma samples.

Key words High performance liquid chromatograph (HPI.C), On-line sample pretreatment, Automated

column switching system, Deproteinization, Dilution, Blood plasma, Drug analysis Introduction

HPLC (High Performance Liquid Chromatography) is one of the effective methods for drug testing to monitor the effect of drug and metabolized materials in the field of Pharmacokinetic study. Generally, when HPLC is used to analyze the drug contained in plasma or serum, pretreatment must be done to remove unnecessary compounds such as protein etc. In this treatment, protein has to be precipitated by adding organic solvent and separated by centrifuge. Such a pretreatment involves complicated procedures and a lot of manual handlings which often cause analysis accuracy problems. Therefore, automatization may be required for this process to improve analysis accuracy. _____ This paper was an oral presentation by author at the 7th National Symposium on Drug and Xenobiotic metabolism in Oct 18-22,2003 Nanjing, China Correspondence to Dr Yuki Hashi

Recently, as an improved automation technique of biological sample pretreatment, pretreatment column has been developed to separate protein as impurities. By combining the column switching system, the automatization of pretreatment and improvement of analysis accuracy for sample pretreatment ability are expected.

In this paper, the principle, basic investigation and applications of the newly developed automated column switching HPLC system equipped with the newly developed bio-sample pretreatment column. Shim-pack MAYI-ODS and on-line dilution bypass for drug analysis are discussed.

Experiments

Chemicals and Solvents. Acetonitrile and

methanol were those of HPLC grade purchased from Wako pure chemical, Osaka, Japan. Acetic acid, Sodium acetate trihydrate, phosphoric acid, Sodium dihydrogenphosphate dihydrate, Sodium monohydrogenphosphate. Sodium perchlorate

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monohydrate were those of special grade obtained from Wako Pure Chemical, Osaka, Japan. All standard drugs were also purchased from Wako Pure Chemical, Osaka, Japan. Water was prepared by Milli-QSP.TOC, Japan Millipore Limited.

Equipment. LC-VP, a biological sample analysis system, called Co-Sense for BA, manufactured by Shimadzu, Kyoto, Japan, consisted of LC-10ADvp pump (3 units, two units for gradient elution, one unit for sample loading mobile phase), SIL-10ADvp

autosampler, CTO-10ACvp column oven, SPD-M10Avp photo diode detector, DGU-14A degasser unit, FCV-12AH high pressure flow switching valve and SCL-10Avp system controller. This system was controlled by CLASS-VP workstation software. Shim-pack VP-ODS (150 mm x 4.6 mm i.d.) and Shim-pack MAYI-OD (10 mm x 4.6 mm i.d.) (Shimadzu, Kyoto, Japan) was used for separation column and sample pretreatment column, respectively.

Fig 1 Flow diagram of the HPLC system 1.Degas unit 2. Pump 3.Autosampler 4.On-line sample dilution bypass 5.Mixer 6.High pressure flow switching valve 7.Pretreatment column 8.Analytical column 9.Column oven 10.Detector M1, M2: Mobile phase M3: Sample loading mobile phase

Fig 2 Extraction of drug components from plasma bymeans of a Shim-pack MAYI-ODS column

Result and Discussion

Effect of pretreatment column. Since protein in blood plasma and serum causes deterioration and clogging to separation column, it should he removed during the pretreatment process. The newly developed Shim-pack MAY1-ODS is an internal reverse phase column coated with hydrophilic polymer. Large molecules such as protein are prevented from entering the micro pores inside of silica gel, while small molecules such as drugs are able to penetrate into micro pores and are retained by stationary phase. Large molecules are separated from

the system, and drugs trapped by the pretreatment column are concentrated and eluted by mobile phase to analytical column.

The effect of deproteinization by Shim-pack MAYI-ODS pretreatment column and analytical conditions are shown in Fig. 3 and Table 1, respectively. Isopropylantipyrine was added to plasma for the experiment evaluation, As shown in the lower chromatogram of Fig.3, protein (peak 1) is trapped and eluted out within the first 3 minutes completely. After protein was removed from the system, the drug was injected to analytical column by changing the flow line position of the high pressure

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6-port valve. As a result, upper chromatogram in Fig.3 was obtained (peak 2; isopropylanitipyrine).

Table 1. Analytical conditions for determination of

isopropylantipyrine For sample injection

Pretreatment column Shim-pack MAYI-ODS (10 mm x 4.6 mm i.d.)

Mobile phase A: 0.1% phosphoric acid B: acetonitrile A/B=95/5 (V/V) Flow rate 2.0mL/min Dilution factor 8 For separation

Column Shim-pack VP-ODS (150 mm x 4.6 mm i.d.)

Mobile phase A: water B: acetonitrile A→B (5%→95%) Flow rate 1.0mL·min-1 Temperature 40℃ Detection 275nm: isopropylantipyrine

280nm: plasma protein (SPD-M10AVP)

Effect of On-line Dilution Bypass. Most of the

drugs are bound with protein in plasma or serum. During deproteinization, there is a possibility that

drugs may be lost in some degree, which causes low recovery rate of drugs. Proteins are bound with drugs due to ion attraction or hydrophobic interaction. To weaken these bindings between protein and drugs, optimization of analytical conditions, such as change of pH or of ionic strength and the addition of organic solvent, may be required. Therefore, the sample loaded mobile phase of Co-Sense for BA was selected to weaken those interactions. In addition, an on-line dilution bypass was designed for smooth injection of large volume plasma or serum sample. Using this on-line dilution function, the injected sample was diluted by eight times automatically.

The chromatogram to evaluate the effect of on-line dilution bypass on the recovery rate of indometacin is shown in Fig.4. Indometacin has a strong binding affinity to protein so it was selected and added to plasma for the experiment evaluation.

The analytical conditions are shown in Table 2. The sample was prepared by spiking indometacin standard into plasma. When 500μL of the plasma sample spiked by indometacin was injected, the recovery rate was 50% without dilution bypass and significantly increased to almost 100% with the use of online dilution bypass. The result with various injection volumes from 100µL, 200µL to 500µL is shown in Fig 5.

Fig 3 Effect of deproteinization by Shim-pack MAYI-ODS column

Fig 4 Effect of on-line dilution bypass on the recovery of drugs

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The peak response was increased according to injection volume. The shape of peak was not changed even at 500μL of injection volume. This result

indicated that, under large sample volume injection, the drug in plasma could be concentrated and recovered from protein.

Fig 5 Injection volume and shape of peak

Fig 6 Linearity of peaks of phenytoin and carbamazepine in plasma


idometacin For sample injection


Mobile phase A: 0.1% phosphoric acid B: acetonitrile A/B=95/5 (V/V) Flow rate 2.0mL·min-1 Dilution factor 8 For separation


Mobile phase A: 0.1%trifluoroacetic acid

B: acetonitrile containing 0.1% trifluoroacetic acid

A→B (5%→95%) Flow rate 1.0mL·min-1 Temperature 40℃ Detection 315nm: (SPD-M10AVP)

Linearity and reproducibility. For the

evaluation of drug linearity in spiked plasma sample, phenytoin and carbamazepine was added to plasma to be a final concentration of 0.2mg-1, 1mg-1, 4mg-1 and 20mg-1. The linearity of peaks obtained from this system and the recovery of phenytoin and carbamazepine are shown in Fig 6 and Table 3, respectively.

Table 3 Recovery of phenytoin and carbamazepine Concentration

(mg·L -1) Phenytoin Carbamazepine

20 98% 95% 4 98% 96% 1 100%< 100%< 0.2 92% 94%

The analytical conditions are shown in Table 4. In addition, the chromatograms of phenytoin and carbamazepine in plasma are shown in Fig 7 and Fig

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8, respectively. It takes 20 minutes to complete the analysis including the column cleaning and the

initializing of gradient elution. Within this

concentration range, the excellent linearity and recovery rate of drugs were obtained.

Fig 7 Chromatogram of phenytoin in plasma (210nm) Upper: 0.2mg·L-1 spiked, 50µL injected; Lower: 2mg·L-1 spiked, 50µL injected

Fig 8 Chromatogram of carbamazepine in plasma (300nm) Upper: 0.1mg·L-1 spiked, 50µL injected; Lower: 1mg·L-1 spiked, 50µL injected


phenytoin and carbamazepine For sample injection


Mobile phase A: 100mM acetate (Na) buffer (PH=4.7)

B: acetonitrile A/B=95/5 (V/V) Flow rate 2.0mL·min-1 Dilution factor 8 For separation Column Shim-pack FC-ODS

(75 mm x 4.6 mm i.d.) Mobile phase A: 20mM phosphate (Na)

buffer (PH=2.5) B: methanol A→B (50%→85%) Flow rate 1.0mL·min-1 Temperature 40℃ Detection 210nm: phenytoin

300nm: carbamazepine (SPD-M10AVP)

Table 5 Reproducibility of peak area of phenytoin and carbamazepine (1mg/μL each, 50μL injected)

Phenytoin (μAbs.sec)

Carbamazepine (μAbs.sec)

No.1 No.2 No.3 No.4 No.5

238911 240042 237208 238317 239678

98548 98367 98706 98481 98152

Average 238832 98451 Standard deviation (s)

1127.9 207.3

Coefficient of variation (%)

0.472 0.211

In addition, the reproducibility of peak area of

phenytoin and carbamazepine in plasma sample is shown in Table 5. In this table, the plasma sample was prepared by spiking each drug to be 1mg/L of concentration and was injected with 50μL, 5 times to HPLC. This automated sample pretreatment system provided excellent reproducibility due to less human errors as compared to manual sample preparation.

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Suppression of carry over For improvement of analysis accuracy, it is very important to suppress the carry over since the concentration of dispensed blood sample is significantly different from one of non-dispensed blood sample.

Table 6 Carryover of drug standards

Condition A"I)

Condition B*2)

Diazepam"3) 0.09% 0.09% Phenytoin "3) 0.04% 0.02% Carbamazepine"3) 0.04% 0.05%

Phenylbutazone"3) 0.28% 0.14% Reserpine"3) 0.08% 0.03% Ketoprofen"3) 0.05% 0.03% Warfarin"3) 0.03% 0.02% Naproxen"3) 0.19% 0.11%

"I): mobile phase for sample injection: I00mM acetate (Na) buffer (pH=4.7); *2): mobile phase for sample injection: 100mM acetate (Na) buffer (pH=4.7) /acetonitrile=9/1 (v/v); "3): 200 mg/L standard solution; "4): 10 mg/L standard solution A concentration of less than 10% of the organic solvent was used for sample loading mobile phase to prevent the precipitation of protein. The absorption of hydrophobic drugs to sample injection flow line was not observed.

Table 7. Analytical conditions for determination of drugs in plasma

For sample injection


Mobile phase A: 100mM phosphate (Na) buffer (PH=2.1 or 6.8)

B: acetonitrile A/B=100/0～90/10 (V/V) Flow rate 2.0mL·min-1 Dilution factor 8 For separation


Mobile phase A: buffer solution B: methanol A→B (15%→85%) Flow rate 1.0mL·min-1 Temperature 40℃

Detection 205, 235, 250, 280 nm, (SPD-M10AVP)

In this paper, to suppress the carry over, the materials of flow line used for autosampler was investigated. In case of sampling needle, a special metal was coated on its surface. PEEK resin was used for the seal materials of the needle and switch valve. The effect of suppression of carry over is shown in Table 6.

Table 8 Types of mobile phase and retention times of drugs

Mobile phase (1) (2) (3) Lidocaine 13.3 - 15.0 Noscapine 14.7 - 19.6 Chlopheniramine 15.6 21.2 16.6 Propranolol 16.9 20.7 16.8 Diphenhydramine 17.2 22.1 17.1 Phenytoin 17.2 17.0 - Isopropylantipyrine 17.7 17.7 17.8 Chlopropamide 17.8 15.2 17.7 Verapamil 17.8 21.9 17.9 Carbamazepine 17.8 17.8 - Acetohexamide 18.3 15.7 19.2 Reserpine 18.6 22.7 - Imipramine 18.8 24.4 18.9 Nifedipine 18.7 19.0 - Ketoprofen 19.5 16.7 18.7 Naproxen 20.1 16.4 - Diazepam 20.2 20.2 - Warfarin 20.5 15.3 19.3 Phenylbutazone 21.0 17.2 - Ibuprofen 22.2 19.2 21.3

(Unit: minute) (1): 20 mM phosphate (Na) buffer (pH=2.5) including 100mM sodium perchlorate; (2): 20mM phosphate (Na) buffer (pH=6.9); (3): 100mM acetate (Na) buffer (pH=4.7)

Determination of analytical conditions. After 3 min of sample injection, the high pressure valve was switched, and the gradient elution was started by use of buffer and methanol (from 15 % to 85 %). The plasma sample spiked by each drug was prepared to be 5 mg/L as each drug concentration. Analytical conditions and observed retention time of each drug were shown in Table 7 and Table respectively. In this experiment, 30 minutes were sufficient to complete the analysis including the column cleaning and initializing of gradient elution. In order to retain all the drugs effectively using pretreatment column, acidic pH phosphate buffer was selected for acidic drugs, on the other hand, neutral

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pH phosphate buffer was selected for basic drugs. As an acidic pH buffer, acetate buffer can be also selected as an alternative sample loading mobile phase. If the basic drugs can be retained by the pretreatment column, acidic buffer such as acetate buffer may be also used. In case of acidic drugs, if pH value of sample loading mobile phase is increased, ion attraction between protein and drugs is getting strong. As a result, the recovery rate of drug may be

reduced, in case of low concentration of sample loading mobile phase or large volume injection, especially. In case of hydrophobic drugs, these drugs can be still retained by the pretreatment column under the analytical conditions including organic solvent. Since organic solvent can weaken the hydrophobic interaction to protein, improvement of drug recovery rate may be expected.

Fig 9 Chromatogram of 8 drugs in plasma (205nm) Upper: spiked with 1mg·L-1 of each drug, 250µL injected Lower: No spiked, 250µL injected



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As a separation mobile phase, in case of acidic drugs, the elution time with neutral mobile phase is faster than one with acidic mobile phase as shown in Table 8. The selection of mobile phase is based on separation of impurities derived from plasma and analysis time. When acidic mobile phase was selected for elution as shown in Fig. 9 –12, it was relatively easy to separate the target drugs, which were eluted within 19 min, from plasma matrix compounds. In case of acidic drugs and hydrophobic drugs which were eluded more than 19 min, the separation of those drugs were improved by adding acetonitrile in

the sample loading mobile phase or by changing initial concentration of acetonitrile from 50 % to 70 % in gradient elution (refer to Table 9 and Fig. 13). In addition, in case of acidic drug separation, neutral mobile phase may be selected to elute these acidic drugs faster. As a result this may be an alternative approach to improve the separation of these drugs from plasma matrix. The recovery of acidic drugs in plasma is shown in Table 10. By using on-line dilution bypass function, a stable analysis can be performed even with larger sample injection volume.

Fig 12 Chromatogram of Phenobarbital, phenytoin, and carbamazepine in plasma (250nm)

Upper: spiked with 2mg·L-1 of each drug, 50µL injected; Lower: spiked with 10mg·L-1 of each drug, 50µL injected

Table 9. Analytical conditions for determination of drugs (acidic compounds) in plasma




B: acetonitrile A/B=90/10 (V/V) Flow rate 2.0mL·min-1 Dilution factor 8 For separation


Mobile phase A: 20mM phosphate (Na) buffer (PH=2.5)

B: methanol A→B (50%→70%) Flow rate 1.0mL·min-1 Temperature 40℃ Detection 220nm, 300nm (SPD-M10AVP)

Table 10 Recovery of drugs (acidic compounds) in plasma (2mg·L-1 each)

(1) (2) (3) (4) Cholorpro- pamide

50µL 500µL

94% 100%

- 71%

- -

Ketoprofen

50µL 250µL 500µL

98% 97% 96%

- - 54%

97& 96& -

Naproxen

50µL 250µL 500µL

100% 90% 83%

- - 21%

98% 95% -

Warfarin

50µL 250µL 500µL

100% 100% 99%

- - 60%

98% 96% -

Ibuprofen 50µL 500µL

99% 100%

- 57%

- -

(1): Injection Volume；(2): Recovery(%) Condition A*)； (3): Recovery(% ) Condition A*) (no bypass); (4): Recovery(%) Condition B**); *): mobile phase for sample injection: 100mM acetate (Na) buffer (pH=4.7)/acetonitriIe-9/l (v/v); **):mobile phase for sample injection: 100mM phosphate (Na) buffer (pH=2.l)/acetonilrile=9/l (v/v)

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Fig 13 Chromatogram of 6 drugs in plasma(220nm, 300nm) Upper: spiked with 2mg·L-1 of each drug, 100µL injected; Lower: 2mg·L-1 of standard drug, 100µL injected

Fig 14 Chromatogram of diazepam in plasma (312nm) Upper: 0.2mg·L-1 spiked, 50µL injected Lower: 2mg·L-1 spiked, 50µL injected

Fig 15 Chromatogram of reserpine in plasma (300nm) Upper: 0.1mg·L-1 spiked, 50µL injected; Lower: 1mg·L-1 spiked, 50µL injected

Fig 16 Chromatogram of phenylbutazone in plasma (300nm) Upper: 4mg·L-1 spiked, 50µL injected; Lower: 4mg·L-1 of the standard drug, 50µL injected

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Fig 17 Chromatogram of warfarin in plasma (315nm) Upper: 0.1mg·L-1spiked, 50µL injected; Lower: 1mg·L-1 spiked, 50µL injected

Fig 18 Chromatogram of naproxen in plasma (330nm) Upper: 0.1mg·L-1 spiked, 50µL injected; Lower: 1mg·L-1 spiked, 50µL injected

Application Several analytical conditions

described in section "Determination of analytical conditions" were used for each drug analysis. Acetate buffer was selected for sample loading mobile phase. In order to avoid choke of flow line caused by protein precipitation, less than 10 % of the organic solvent was added to mobile phase. To shorten the analysis time, Shim-pack FC-ODS (Shimadzu, Kyoto, Japan) was selected due to its high efficiency and high performance. As described in section "Determination of analytical conditions" about the elution index using acidic and neutral mobile phase, mobile phase was selected based on its pH value that the target drug in plasma can be easily separated from impurities derived from plasma itself. The condition of gradient was considered based on the analysis time and the separation. If peak tailing was observed, the shape of peak could be improved by adding sodium perchlorate to mobile phase. Photo Diode Array detector was used to select the wavelength to obtain well separated chromatogram.

Table 11 Analytical conditions for diazepam For sample injection




Column Shim-pack FC-ODS (75mm x 4.6 mm i.d.)


B: methanol A→B (60%→85%) Flow rate 1.0mL·min-1 Temperature 40℃ Detection 312nm (SPD-M10AVP)

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Table 12 Analytical conditions for determination of reserpine






Mobile phase

A: 20mM phosphate (Na) buffer (PH=2.5) 100mM sodium perchlorate

B: methanol A→B (30%→85%) Flow rate 1.0mL·min-1 Temperature 40℃ Detection 300nm (SPD-M10AVP) Table 13 Analytical conditions for determination of

phenylbutazone For sample injection





Mobile phase



Table 14 Analytical conditions for determination of warfarin








Table 15 Analytical conditions for determination of naproxene



Mobile phase A: 0.1% phosphoric acid B: acetonitrile A/B=95/5 (V/V) Flow rate 2.0mL·min-1 Dilution factor 8 For separation


Mobile phase


B: methanol A→B (60%→85%) Flow rate 1.0mL·min-1 Temperature 40℃

Detection 300nm (SPD-M10AVP)

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The several application results using this system as well as the analytical conditions are shown in Fig. 14 - 18 and Table 11 - 15, respectively. As shown in Table 16, good recovery rate of drugs was obtained from each drug analysis. The analysis time including column washing and initializing of gradient elution is 20 minutes (Fig 14, 17 and 18) and 23 minutes (Fig 15 and 16).

The separation of drugs from plasma impurities should be carefully studied. Longer wavelength results in better selectivity than shorter one when UV-VIS detector is selected. If the selectivity for sample detection cannot be obtained, selection of detector such as florescent or MS, etc. needs to be considered.

Table 16 Recovery of drugs in plasma Concentration Recovery (%)

Diazepam 2mg·L-1 0.2m·L-1

94% 100%<

Reserpine 1 mg·L-1 0.1 mg·L-1

100%< 100%<

Phenylbutazone 4 mg·L-1 99%

Warfarin 0.1 mg·L-1 1 mg·L-1

92% 99%

Naproxen 0.1 mg·L-1 1 mg·L-1

98% 100%<

Conclusion

An essential sample pretreatment such as

deproteinization and dilution can be eliminated by

using automated pretreatment HPLC system. Therefore, this process is automatically operated with the following advantages. 1) pretreatment process can be totally omitted, 2) high throughput of sample treatment is improved and 3) target drugs can be recovered with high accuracy and precision.

Although the optimization of sample loading mobile phase, analytical column, mobile phase, detection wavelength, gradient condition, etc is time consuming, if several analytical conditions such as sample loading mobile phase and mobile phase are established, optimization of analytical conditions can he easily determined by this system.

Acknowledgment The authors wish to thank the

following for valuable advice and technical assistance to develop the Co-sense for BA system and Shim-pack MAYI-ODS column; Dr. Naoki Asakawa, Mr. Ei-ichi Yamamoto, Mr. Kaoru Murata and Mr. Yasushi Ishihama (Eizai). References 1 Hagetstam IH, Pinkerton TC, Internal surface reversed-phase

silica supports for liquid chromatography. Anal. Chcm.1985; 57: 1757-1763.

2 Haginaka J, Yasuda N, Wakai J, Matsunaga H, Yasuda H and Kimura Y, Internal-surface reversed-phase silica support for direct injection determination of drugs in biological fluids by liquid chromatography. Anal Chem. 1989; 61(21): 2445-2448.

3 Yamamoto E, Murata K, Ishihama Y, Asakawa N, Methylcellulose-immobilized reversed-phase precolumn for direct analysis of drugs in plasma by HPLC. Anal. Sci. 2001; 17(10): 1155-1159.

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