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BIOEQUIVALENCE ESTIMATION BASED ON

PEAK AREAS OF UNKNOWN METABOLITES

VALENTINA ANUTA1,2

*, A. ALDEA3, OLIMPIA NEAGU

4,

I. MIRCIOIU1, DALIA MIRON

1,2, F. RADULESCU

1,2,

MONICA SOARE-RADA1, F. ENACHE5 

1 Biopharmacy&Pharmacol Res, 23 Pitar Mos Str., Bucharest, Romania

2 UMF Carol Davila, Faculty of Pharmacy, 6 Traian Vuia Str.,

 Bucharest, Romania3Pfizer Romania S.R.L., Bucharest, Romania

4 National Medicine Agency, Bucharest, Romania

5Petru Poni Technical College., Onesti, Romania

*corresponding author: office@bpres.ro

Abstract Te aim of this study was the evaluation of some specific bioanalytical

approaches for determination of metabolites pharmacokinetics in bioequivalence studies.Particularly it is considered the possibility of evaluation of bioequivalence in

case of lack of standards for metabolites.

Liquid-liquid extraction of plasma samples followed by reversed-phase

mechanism separation and flourescence detection was chosen for the determination of tramadol and its active metabolite O-desmethyl-tramadol in plasma samples. The internal

standard was propranolol. HPLC method used a Kromasil 100-5 C18 column, with a

gradient elution with a flow rate of 1 mL/min, at 30°C column temperature. 24 healthy

volunteers were enrolled into a single-dose (100 mg), two-way, cross-over bioequivalence

study. Method 1 considered only metabolite’s peak area in pharmacokinetic evaluation of 

the products. Method 2 used the ratio between the metabolite’s peak area and internalstandard’s peak area in pharmacokinetic evaluation. The third method was the usual

method used in evaluation of pharmacokinetics – based on concentrations. The results of 

the three methods for evaluating pharmacokinetics and bioequivalence were compared.For both alternative methods the estimations concerning 90% confidence

intervals for ratios of pharmacokinetic parameters defining bioequivalence were quite

similar to the standard method.The proposed alternative screening methods, more rapid and simple, can give

useful information about bioequivalence, variability in pharmacokinetics associated with

formulation and help fenotyping of subjects or identifying outliers.

Rezumat

Studiul prezintă evaluarea a două abordări bioanalitice „alternative” pentrudeterminarea farmacocineticii metaboliţilor, aplicabile atunci când lipsesc standardele de

metaboliţi şi de evaluarea farmacocinetică comparativă a produselor  în cadrul studiilor de

 bioechivalenţă. 

Pentru determinarea tramadolului şi a metabolitului său O desmetil-tramadol din probele de plasmă s-a utilizat un mecanism de separare cu fază inversă şi detecţie în

fluorescenţă, prepararea probelor fiind făcută  prin extracţie lichid-lichid. Standardul intern

ales a fost propranololul. Separarea cromatografică s-a realizat pe o coloană de tip C18,

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FARMACIA, 2007, Vol.LV, 6 681

(Kromasil 100-5 C18), eluţia fiind de tip gradient, la temperatura de 30°C. Metoda

cromatografică a fost aplicată în cadrul unui studiu de bioechvalenţă de tip încrucişat, cu

două perioade şi două secvenţe, pe 24 de voluntari sănătoşi cărora li s-a administrat o doză

unică de Tramadol (100mg).

Evaluarea rezultatelor în cazul O-desmetil-tramadolului s-a realizat prin maimulte metode:

Metoda 1 – a considerat, în evaluarea farmacocineticii produselor, aria peak-uluide metabolit.

Metoda 2 – a considerat, în evaluarea farmacocineticii produselor, raportul dintre aria

 peak-ului de metabolit şi aria peak-ului de standard intern.

Metoda 3 (metoda uzuală de evaluare a farmacocineticii în studiile de bioechivalenţă) -s-a bazat pe calcule plecând de la concentraţia efectivă a metabolitului.

Estimările intervalului de încredere 90% pentru rapoartele parametrilor 

farmacocinetici definitorii ai bioechivalenţei prin cele două metode alternative au fost

similare cu cele furnizate de metoda standard.

Metodele alternative de screening propuse, mai rapide şi mai simple decât

metoda uzuală, pot oferi informaţii în legătură cu bioechivalenţa, variabilitateafarmacocineticii ca efect al formulării şi ajută la fenotiparea subiecţilor . 

- pharmacokinetic validation - metabolites in bioequivalence

INTRODUCTION

The increasing number of metabolites required to be evaluated 

concerning their pharmacokinetics in drug evaluation as well as their low

concentrations in plasma, rise specific, new, complex issues both in

 bioanalytical assay and biostatistical estimation of bioequivalence of drugs

with active or inactive but highly variable metabolites.

Tramadol is rapidly and almost completely absorbed after oral

administration but its absolute bioavailability is only 65–70% due to first-

 pass metabolism [1].The biotransformation of tramadol in human subjects has been

shown to be carried out by the isoenzyme cytochrome P4502D6 (CYP2D6).

Since CYP2D6 mediated metabolization is largely variable, deciding

 bioequivalence based on parent drug or both on parent drug and metabolite

 plasma levels meets high difficulties first of all due to a large inter and 

intravariability in kinetics and extent of metabolization.

The main metabolites have been found to be N -desmethyltramadol

and  O-desmethyltramadol (OD -Tramadol), of which only the latter is

 pharmacologically active [2].

Although maximum plasma concentration for the metabolite is 2-5

times lower than for tramadol (100–300 ng/ml [3]), OD-Tramadol presents a10-times higher affinity for the μ-opioid receptors, having an important

contribution to analgesic effect of the drug.

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Figure 1

Phase I metabolism of Tramadol

The present paper refers to an analytical experimental and 

simulation calculus in a bioequivalence study. Tramadol has more than three

metabolites quantifiable in plasma by HPLC with fluorescence detection

[4]. We tried to estimate the bioequivalence of two formulations containing

tramadol as active substance, based on metabolite considered first known

and then as unknown. Bioequivalence was estimated, based on parent drug

and OD-Tramadol.

It was considered post factum an analitycal, pharmacokinetic and 

 biostatistic evaluation starting from an “unknown peak” in plasma samples,

absent in plasma blanks, increasing and decreasing in real samples,suggesting “pharmacokinetic-like profile”. The comparison of the obtained 

results revealed such high similarity of bioequivalence confidence intervals

that finally appear that, at least in this case, all problems connected with

identification and estimation of the concentrations of metabolite can be

avoided at a first-screening evaluation.

MATERIALS AND METHODS

Reagents Chemicals used were of HPLC grade: acetonitrile and methanol were

 purchased from Lab Scan, trifluoroacetic acid, methylen chloride, sodium

hydrogen carbonte and hydrochloric acid were manufactured by Merck.

HPLC grade water purified by a TKA-Genpure UV system was

used for both chromatographic elution and samples preparation.

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HPLC conditions The analyses were carried out using a Waters 600 Multisolvent

Delivery System, a Waters 717 plus autosampler and a HP 1046A

Fluorescence Detector, operating at λ ex=277 nm and λ em=311 nm.

The separation was performed on a Kromasil 100-5-C18, Azko

 Nobel, 150*4.6 mm column at 30°C. The mobile phase consisted in agradient of 0.1% trifluoroacetic acid (A) and acetonitrile (B) (table I).

Table IMobile phase composition for determination of tramadol and OD-Tramadol 

Time (min) A (%) B (%)

0 75 25

1.00 75 25

1.01 65 35

4.5 65 35

4.51 75 25The flow-rate was 1mL/min, and the injection volume was 100 μL.

Standard and plasma solutions

Stock solutions of tramadol, OD-tramadol and internal standard 

(propranolol) in concentrations of 50 µg/mL were prepared separately by

dissolving 5 mg of each substance in methanol.

Separate solutions in the range 5-500 ng/mL for Tramadol and 2.5-

250 ng/mL for O-Desmethyl-Tramadol were prepared for the calibration

curves and quality control samples.

The working internal standard solution (2.5 μg/mL) was prepared

in methanol and stored at 4°C during the study.Preparation of spiked calibration standards and QC (quality

control) samples

The plasma samples were stored in the freezer at −20°C and 

allowed to thaw at room temperature before processing.

To 0.5 mL of plasma sample 50 μL Internal Standard (Propranolol

2.5 μg/mL in methanol), 100 μL 0.5M sodium hydrogen carbonate solution

and 3 mL methylene chloride were added. The resulting samples were

shaken horizontally for 20 minutes at 120 rpm, and 2.5 mL from the lower 

organic layer were transferred into another test tube and evaporated to

dryness, at 40°C under a nitrogen stream. The sample was reconstituted in

300 µl of 0.1M hydrochloric acid.

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Clinical Study

The study protocol was approved by the National Medicine Agency

and the Independent Ethics Committee.

The study was projected as a single-dose, randomized, two-

treatments, two-periods, two-sequences cross-over study under fasting

conditions, comparing equal doses of test and reference products, with sixdays washout interval between Period I and Period II dosing.

24 male and female healthy volunteers received a single dose of 

100 mg tramadol.

Venous blood samples (approximately 5mL) were collected 

through a catheter inserted in an antecubital vein, pre-dose (0 hours) and at

0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0, 5.0, 6.0, 8.0, 10.0, 12.0 and 24.0 after the

drug administration.

Data Evaluation Methods

After the bioanalytical assay, the data were evaluated using three

alternative methods:

Method 1 – used the metabolite peak area as a “surrogate

concentration”.

Method 2 – used the ratio between the metabolite’s peak area and 

internal standard peak’s area as a “relative concentration”.

Method 3 - used concentrations (calculated on calibration curves)

versus sampling times.

Evaluation of the pharmacokinetics of tramadol and O-Desmethyl-

Tramadol and estimation of 90% confidence intervals for pharmacokinetic

 parameters defining bioequivalence were further calculated starting from the

described three types of data.

RESULTS AND DISCUSSION

1. Bioanalytical study

HPLC method:Typical chromatograms of drug free and plasma from a volunteer 1

hour after the oral ingestion of 100 mg tramadol are presented in figure 2:

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    m     V

-100.00

0.00

100.00

200.00

300.00

400.00

500.00

600.00

700.00

800.00

Minutes

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10. 00 11.00 12.00

 

    m     V

-100.00

0.00

100.00

200.00

300.00

400.00

500.00

600.00

700.00

800.00

Minutes

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 1

     O     D     T    r    a    m    a     d    o     l

     T    r    a    m    a     d    o     l

     S     I

 A B

Figure 2 Chromatogram of drug free plasma sample (A) and plasma from a volunteer 1 hour 

after the oral ingestion of 100 mg tramadol (B)

It can be observed that besides the peaks for drug and internal

standard, another significant peak appears which was considered a peak of a

metabolite.

Validation of the bioanalytical method:

The calibration curve for OD-Tramadol was linear in the range 2.5-

250 ng/mL (r=0.9993) (Figure 3).

Figure 3Calibration Curve for OD-Tramadol

The lower limit of quantitation (LLOQ) was 2.5 ng/ml ( N = 6).

Both intra-day and inter-day accuracy and precision were situated within the accepted limits. The precision was better than 3% and the

deviation from nominal concentration did not exceed 10 % at all levels.

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The mean recovery of OD-Tramadol from plasma samples was

77% (range 74-80% for three different concentration levels).

The stability of unprocessed plasma samples was studied for 3

months at the storage temperature (-20°C), for 30 hours at room

temperature, and after three freeze and thaw cycles. The concentration

changes relatively to the nominal concentration were less than 15%,indicating no significant substance loss during the study.

The processed plasma samples proved to be stable for at least 66

hours.

2. Pharmacokinetic results

Obtained data were used for pharmacokinetic and biostatistical

evaluations.

Pharmacokinetic parameters obtained by standard method are

 presented in table II.

Table IIPharmacokinetic parameters of OD-Tramadol

after oral administration of 100 mg Tramadol to 24 healthy volunteers

AUC 0-∞ (ng/ml*h) AUC 0-n (ng/ml*h) C max (ng/ml)

R T R T R T

Mean 988.19 1024.9 904.23 930.81 89.833 92.375

SD 415.42 403.63 389.86 391.56 43.682 45.522

Median 1045.2 1078.8 972.13 1006.8 92.5 101

Min 109.02 183.4 85 128.75 16 16

Max 1636.8 1611.6 1529.5 1530.5 194 174

AUC0-n - area under curve in the time interval 0-nAUC0-∞ - area under curve in the time interval 0-∞ C max – maximum concentration

The literature provides substantial evidence for the relationship

 between genetic polymorphism of CYP2D6 and changes in the

 pharmacokinetics of tramadol and its metabolite [5, 6, 7]. Our results are

consistent with literature data and suggest a bimodal distribution for C max 

and AUC0-∞, as can be seen in figure 4.

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FARMACIA, 2007, Vol.LV, 6 687

A BFigure 4

Frequency for distribution of Cmax (A) and AUC0-∞ (B) for OD-Tramadol

The mean curve plasma levels vs. time were similar whatever the

use of concentrations, peak areas or Analyte/Internal standard ratios as

measures of plasma levels (Figure 5).

0 6 12 18 24 T(h)

0

200000

400000

600000

800000

1000000

1200000

1400000

  M  e  a  n  o  f  A  r  e  a  (  A  U  )

MeanCurve Treatement T- Treatement R

GroupName:RGroupName:T

 

0 6 12 18 24

 T(h)

0

0.1

0.2

0.3

0.4

  M  e  a  n  o  f  A  r  e  a  r  a  t  i  o  (  A  r  e  a  O  D  T  /  A  r  e  a  I  S  ) MeanCurveTreatementT- TreatementR

GroupName:RGroupName:T

 

0 6 12 18 24

 T(h)

0

30

60

90

  M  e  a  n  C  o  n  c  (  n  g  /  m  L  )

MeanCurveTreatementT- TreatementR

GroupName:RGroupName:T

 

Figure 5Mean plasma levels for OD-Tramadol after oral administration of 100 mg

Tramadol to 24 healthy volunteers

Estimation of pharmacokinetic parameters and bioequivalence

based only on peak areas:

Evaluation of intensive pharmacokinetic parameters for OD-

Tramadol lead to comparable results for all three methods, “relative results”

Mean curve Peak Area vs. Time Mean curve Ratio Peak 

Analyte Area/IS Area vs. Time

Mean curve Concentration

vs. Time

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FARMACIA, 2007, Vol.LV, 6688

not being influenced by the lack of metabolites standards, as can be seen

from figure 6:

Figure 6 

Intensive pharmacokinetic parameters for evaluation of OD-Tramadol

Theoretically, since bioequivalence is a comparative analysis, peak 

areas could be taken as source data for calculation in estimation of 

 bioequivalence, the results seem to confirm this hypothesis, as can be seen

from Table III and Figure 7.

Table III90% confidence intervals for ratios of pharmacokinetic parameters AUC 0-∞, AUC 0-n, and C max 

90%

Confidence

interval

AUC 0-∞ 

Analyte area 0.94-1.08

Analyte area /Internal Standard Area 0.95-1.10

Concentration 0.97-1.15

AUC 0-n 

Analyte area 0.93-1.09

Analyte area /Internal Standard Area 0.95-1.10

Concentration 0.96-1.12

C max

Analyte area 0.90-1.11

Analyte area /Internal Standard Area 0.92-1.11

Concentration 0.92-1.11

It is sufficient a superficial examination to see that real data in the

case of OD-Tramadol prove that confidence intervals for the ratio of mean

 pharmacokinetic parameters remain practically the same, whatever the use

or not use of standards (Table III, figure 7).

    T    i   m   e    (    h    )

 

Some intensive pharmacokinetic parameters for OD-tramadol 

0 

2 

4 

6 

8 

10 

12 

14 

16 

Method 1 

Method 2 Method 3 

thalf  Tmax MRT

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Figure 7 90% Confidence interval intervals for ratios of pharmacokinetic parameters

AUC 0-∞, AUC 0-n, and C max for the three methods tested 

CONCLUSIONS

For both alternative methods the estimations concerning 90%

confidence intervals for ratios of means of pharmacokinetic parameters

defining bioequivalence were practically similar to that calculated starting

from the results obtained by standard method.

The proposed methods are, consequently, adequate for metabolites

 pharmacokinetic evaluation for the bioequivalence study.

Although is safer to use standards, since bioequivalence is a

comparative analysis, the use of peak areas could be taken as criterion for 

 prediction of bioequivalence.

Such a method, more rapid and simple to apply for evaluation, can

 be used as a screening method, giving useful information about:-   bioequivalence

-  variability in pharmacokinetics associated with formulation

-  fenotyping of subjects

-  decisions concerning outliers.

REFERENCES

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Pharmacokinet 2004; 43 (13): 879-923

2.  Budd K,  Langford R.- Tramadol revisited.-  Br J Anaesth.  1999Apr;82(4):493-5

Cmax

AUC0-n

AUC0-∞ 

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3.  Lewis KS, Han NH.- Tramadol: a new centrally acting analgesic.-  Am

 J Health Syst Pharm. 1997 Mar 15;54(6):643-52

4.  Ardakani YH, Rouini MR.- Improved liquid chromatographic

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hypoalgesic effect of tramadol in relation to CYP2D6- Clin

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6.  Gan SH, Ismail R, Wan Adnan WA, Zulmi W.- Impact of CYP2D6

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