Technical Note

CHROMATOGRAPHY, Vol.31 No.3 (2010)

Technical NoteSimple Method for Determining Mitotane Concentration in
Human Blood PlasmaMasako Nakazawa1), Mari Egawa2), Toshio Tanaka2), and Ritsuo Aiyama1)
1)

Yakult Honsha Co. Ltd., Medical Development Department, 1−12−5 Owada−machi, Hachioji, Tokyo 192−0045, Japan
2)

Shimadzu Techno−Research, Inc., 2−13, Nishinokyo−Sanjyobocho, Nakagyo−ku, Kyoto 604−8435, JapanAbstract
We developed a simple method for determining mitotane concentration in a patient’s plasma using a conventional HPLC instrument equippedwith an ultraviolet−visible photometer and a stainless steel column packed with octadecylsilanized silica gel. The method was validated interms of specificity, linearity, precision, accuracy, quantitation limit, and stability of test solutions in the concentration range of 0.2 to 40 µg/mL of mitotane in plasma samples.
Keywords : mitotane, Opeprim, plasma concentration, dichlorodiphenyldichloroethane, HPLC, validation

reduced until the maximum tolerated dose is achieved, and theIntroduction
Mitotane (o,p′−DDD [o,p′−dichlorodiphenyldichloroeth-plasma concentration of mitotane should be immediatelyane], Fig. 1) is a derivative of the insecticide dichlorodiphenyl-determined [6]. The use of gas chromatography for determining thetrichloroethane that specifically inhibits tumor cells of the adrenal

plasma concentration of mitotane has been reported [5−7].

cortex and its production of hormones. The encapsulated

However, a conventional high−performance liquid chromatography

formulation of mitotane is known as Opeprim (500 mg capsules)(HPLC) system is commonly used in hospitals to determine plasmaand is used to treat adrenocortical tumors [1−4]; it causes central

concentrations of mitotane. The objective of this study was tonervous system damage but no bone marrow depression. In thedevelop and validate a simple assay that can be used with arecommended treatment schedule, the patient begins at 1 to 2

conventional HPLC at any hospital to determine plasma mitotane

capsules of Opeprim, 3 times per day, and the dose is increasedconcentration.

incrementally up to the effective dose. The therapeutic plasma
Materials

concentration range of mitotane in patient was reported as 14−20µg/mL [5]. If severe side effects appear, the dose should be

Our conventional HPLC system was composed of aShimadzu SCL−10Avp system controller, an LC−10ADvp solvent
delivery unit, an SIL−10A autosampler, a CTO−10ACvp column
oven, a DGU−14A online degasser, an SPD−10AVvp ultraviolet−
visible absorption detector, and CLASS−VP data−processing
software on a personal computer. A stainless steel column packed
with octadecylsilanized silica gel (Inertsil ODS−3, 4.6 mm insidediameter, 25 cm length, 5 µm particle diameter) was purchasedfrom GL Sciences, Inc.
Several lots of standard human plasma with added

Fig. 1 Chemical Structure of Mitotane
Correspondence : Masako Nakazawa―１０３―

CHROMATOGRAPHY, Vol.31 No.3 (2010)

anticoagulant (sodium heparin) were supplied by Cosmo Bio Co.

a final volume of 20 mL for each solution, and these solutions wereand were mixed for preventing lot−to−lot variation. The mixed

designated as SD−160 (160 µg/mL), SD−80 (80 µg/mL), SD−40

standard plasma was stored under freezing conditions (below −20(40 µg/mL), and SD−20 (20 µg/mL), respectively. Methanol wasO

added to 8, 4, and 2 mL of STK−2 to a final volume of 20 mL forC) in a Sanyo medical freezer MDF−U536.
Fluka 2,4′−DDD (cat. no. 35485, PESTANAL, analyticaleach solution, and these solutions were designated as SD−8 (8 µg/standard, not less than 99%) was used as the standard mitotanemL), SD−4 (4 µg/mL), and SD−2 (2 µg/mL), respectively. SD−0.8

substance for the determination. Wako p,p′−DDD standard (cat.

(0.8 µg/mL) was prepared from 2 mL of STK−2 and enough

no. 046−27101, for determining residual pesticides) was used as anmethanol to bring the final volume to 50 mL.

internal standard (IS).
Preparation of Internal Standard Solution (IS Solution)

An Ultrafree−MC centrifugal filter device (pore size: 0.45µm; cat. no. UFC 30 HVNB, Millipore Corp.) was used, and theApproximately 10 mg of p,p′−DDD standard, accurately

sample was centrifuged with a centrifugal separator, model CR−15,

weighed, was dissolved in methanol to a final volume of 50 mL (ISsupplied by Hitachi−Koki Co.

stock solution: 200 µg/mL). This solution can be stored at 5 ± 3 OC

A solvent gradient of purified water and methanol was usedfor 4 weeks. The IS stock solution (4 mL) was combined withas a mobile phase. Acetonitrile and methanol for the liquidsufficient methanol to bring the final volume to 20 mL (IS solution:

chromatography were supplied by Kanto Chemical Co. and Wako

40 µg/mL).

Pure Chemical Industries. Purified water with an electricalresistance of at least 18.2 MΩ•cm was prepared with a Milli−Q

Preparation of Solutions for Calibration Curve and BlankUltrapure production unit (Millipore Corp.).

Solutions

Test procedure

acetonitrile, SD solution (or methanol for a blank sample), and IS

Preparation of Standard Stock Solutions (STK−1, STK−2)

solution were added, in this order, according to the blending ratios

Each solution began with 200 µL of blank plasma, to which

To prepare standard stock solution 1 (STK−1, 400 µg/mL),

listed in Table 1. This solution was vortexed and then centrifugedapproximately 20 mg of mitotane standard, accurately weighed,at 10,000 rpm for 3 min. The supernatant was transferred to thewas dissolved in methanol to a final volume of 50 mL. STK−1 (1sample reservoir of a centrifugal filter device and centrifuged atmL) was diluted with methanol to a final volume of 20 mL, and the8000 rpm for 1 min. The filtrates were used as the solutions for a

resulting solution was designated as standard stock solution 2 (STKcalibration curve (CAL solutions) and as blank solutions. The CAL−2, 20 µg/mL). STK−1 can be stored at 5 ± 3 OC for 4 weeks, butsolutions and blank solutions can be stored at 5 OC within 48 hours.

STK−2 should not be stored.
Preparation of Sample Solution
Preparation of Standard Solutions (SD solutions)

Acetonitrile, methanol, and IS solution were added, in thisorder, to 200 µL of the sample plasma. Samples were then vortexed

Methanol was added to 8, 4, 2, and 1 mL of STK−1 to make

Table 1 Volumes of Components of Calibration Curve Solutions (CAL Solutions)
SD Solution

CAL Solution

Blank Plasma(µL)

Acetonitrile
(µL)

(µL)

CAL−4020030050

CAL−2020030050

CAL−1020030050

CAL−520030050

CAL−220030050

IS Solution(µL)

Mitotane Concentration(µg/mL)*SD−1605040SD−805020SD−405010SD−20505SD−8502CAL−1200300

50SD−4501

CAL−0.5200300

50SD−2500.5

CAL−0.2200300

50SD−0.8500.2IS Blank

200

300

50Methanol

500Blank

200

300

100Methanol00* In a 200−µL plasma sample―１０４―

CHROMATOGRAPHY, Vol.31 No.3 (2010)

Masako Nakazawa, Mari Egawa, Toshio Tanaka and Ritsuo Aiyama

and centrifuged at 10,000 rpm for 3 min. The supernatant wasobserved in the chromatogram at 19 min and 21 min, respectivelytransferred to the sample reservoir of a centrifugal filter device and(Fig. 2). No peak appeared in the chromatogram of the blankcentrifuged at 8000 rpm for 1 min. The filtrates were used as thesample at the retention time of mitotane. No difference was

sample solutions and can be stored at 5 OC for 48 hours.

observed between plasma from males and females..

Determination of Plasma Mitotane Concentration

Linearity and Range

Twenty µL of CAL solution or blank solution was injected

A plot of the mitotane concentrations against the ratios of theinto the HPLC instrument, which was operated under themitotane peak area to the IS peak area (8 concentrations) wasconditions described below. For each solution, we then calculated

linear, and correlation coefficients of 0.9998 to 0.9999 were

the ratio of the peak area of mitotane to that of the IS in eachobtained from the results of triple measurements in thechromatogram. A calibration curve was created by plotting theconcentration range of 0.2 to 40 µg/mL (Fig. 3).

peak ratio as a function of the mitotane concentration.
The test was then performed with 20 µL of the sample

Precision and Accuracy

solution, and the peak ratio of mitotane to the IS was calculated in

The relative standard deviations obtained from 5 repeatedthe same manner. The plasma concentration of mitotane can thus

measurements at plasma mitotane concentrations of 0.2, 0.5, 5,

be obtained by the calibration curve method.
Operation Conditions for HPLC
Detector:

UV−VIS photometer (wavelength: 230 nm)

Column:Inertsil ODS−3 (5 µm), 4.6 mm in inside
diameter, 25 cm in length

Guard Column: Guard cartridge column GL Cartridge
Inertsil ODS−3, 5 µm
Mobile Phase A: Purified water
Mobile Phase B: Methanol
Mitotane and IS are determined under an
isocratic condition. After eluting both peaks,
the column is washed and re equilibrated
according to the following table.

Time after Injection(min)

Mobile Phase A(vol %)

Mobile Phase B(vol %)

021.0
21.1
34.0
35.0
45.0
55.0

2020
5550
202080
80
95
95
50
80
80

Flow Rate:

Fig. 2

Typical Chromatogram of Blank Sample and CAL Standard Solution

Fig. 3

Plot of the Ratio of Mitotane Peak Area to IS Peak Area
versus Plasma Mitotane Concentration1.0 mL/min

Column Temperature:

Constant temperature of 40 OC

Sampler Temperature:

Constant temperature of 5 OC

Duration of Measurement:

55 minAnalytical Validation
Specificity
A total of 6 blank plasma samples (2 female, 4 male) were used tovalidate the method. The peaks for the IS and mitotane were

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CHROMATOGRAPHY, Vol.31 No.3 (2010)

and 20 µg/mL ranged between 1.2 and 2.7 %, with accuracies

the residual ratios of mitotane (triplicate runs) were between 99.9

between −0.7 and 2.4 %. The relative standard deviations of the 3−and 102.4 % at the 48−hour sampling point.

day precision test obtained from 3 repeated measurements atplasma mitotane concentrations of 0.5, 5, and 20 µg/mL ranged

Conclusion

between 1.7 and 2.7 %, with accuracies between −2.3 and 3.4 %.

The simple assay method for determining mitotane
concentration in plasma was developed by using a conventional

Recovery Rate

HPLC system equipped with an ultraviolet−visible photometer and

The recovery rates of mitotane and the IS at the concentrations ofa stainless steel column packed with octadecylsilanized silica gel.

both 2 and 20 µg/mL (in triplicate) were greater than 97.4 % for

The method was validated in terms of specificity, linearity,mitotane and greater than 98.7 % for the internal standard.

precision, accuracy, quantitation limit, and stability of test
solutions in the concentration range of 0.2 to 40 µg/mL of mitotane
in plasma samples.

Quantitation Limit
The quantitation limit for mitotane was calculated to be 0.0013 µgfor each injection as the lower limit of quantitation (LLOQ) based

Referenceson the lowest spiking concentration of the analyte in Linearity and[1]Hahner S and Fassnacht M. “Mitotane for adrenocorticalRange (corresponding to 0.2 µg/mL of plasma concentration). The

carcinoma treatment”. Current opinion in investigational

relative standard deviation of triple measurements at thedrugs (London, England : 2000) 6 (4) : 386−394.
[2]concentration was less than 10 %.

Schteingart DE, Motazedi A, Noonan RA and Thompson
NW, Arch Surg., 1982, 117: 1142−1146.

[3]

Stability Test of Sample and Solution
The plasma samples spiked with mitotane (0.5 and 20 µg/mL) were

Boven E, Vermorken JB, van Slooten H, Pinedo HM,Cancer, 1984, 53: 26−29.

Ostable for 4 weeks at −20 C, and the residual ratios of mitotane in[4]

Wooten MD and King DK, Cancer, 1993, 72: 3145−3155.

the plasma samples (triplicate runs) were greater than 94.8 % at the[5]

Terzolo M, Pia A, Berruti A, Osella G, Ali A, Carbone V,

4−week sampling point. When the freeze−thaw cycle was repeatedTesta E, Dogliotti L and Angeli A, J. Clin. Endocrinology &twice, the residual rates of mitotane in the plasma samples

Metabolism, 2000, 85(6) : 2234−2238(triplicate runs) were between 93.6 and 110.6 %. STK−1 was stable[6]

Package Insert of “Opeprim ”, 11 th edition revised on Nov.

[7]

Moolenaar AJ, Niewint JW and Oei IT, Clin. Chim. Acta,O

for 4 weeks at 5 C, and the residual ratios at the 4−week sampling

2009, Yakult Honsha Co., Ltd.

point were greater than 98.3 %. The residual ratio of the IS stock

1977, 76(2) : 213−218.

solution at 4 weeks was greater than 95.5 %. The sample solutionsfor HPLC (0.5 and 20 µg/mL) were stable for 48 hours at 5 OC, and―１
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