Forensic Science International 196 (2010) 93–96

Changes in antidepressant metabolism in pregnancy evidenced by metabolicratios in hair: A novel approach

Lisa O’Brien a,b,*, Carina Baumer c, Detlef Thieme c,d, Hans Sachs c,d, Gideon Koren a,b,e

a Division of Clinical Pharmacology and Toxicology, Hospital for Sick Children, Toronto, Canadab Institute of Medical Science, University of Toronto, Canadac Forensic Toxicological Centre, Munich, Germanyd Institute of Forensic Medicine, Munich, Germanye Department of Pediatrics, Hospital for Sick Children, Toronto, Canada

A R T I C L E I N F O

Article history:

Received 5 June 2009

Accepted 12 August 2009

Available online 8 January 2010

Keywords:

Pregnancy

Antidepressant metabolism

Hair

Segmental analysis

A B S T R A C T

Background: Depression and other psychiatric illnesses are common during pregnancy and are often

treated with antidepressants. Physiological changes of pregnancy may alter the pharmacokinetics of

medications and ultimately affect the dose required to maintain effective therapy. Human hair offers a

safe, non-invasive way to monitor long term systemic exposures to medications.

Objective: To determine whether the ratio of hair antidepressant: major metabolite differed when early

and late pregnancy was compared to the postpartum period.

Methods: Segmental analyses using liquid chromatography–mass spectrometry–mass spectrometry

were performed on hair samples. The mean concentration of parent compound and metabolite was

found for each trimester and the postpartum period.

Results: Twelve women provided hair samples of which nine samples were long enough to analyze the

first and third trimesters along with the postpartum period. Citalopram, venlafaxine, fluoxetine and

sertraline were the antidepressants studied. In the citalopram group, a statistically significant difference

existed between the citalopram:norcitalopram ratio when the first trimester was compared to the

postpartum period (0.89 � 0.26 versus 1.4 � 0.24 respectively, p = 0.022). A statistically significant

difference also existed between the third trimester and the postpartum period for the citalopram group

(0.9 � 0.14 and 1.4 � 0.24 respectively, p = 0.048). No other statistically significant differences were found.

Conclusion: It is important that variations in drug metabolism during pregnancy be considered as these

changes may necessitate a dosage adjustment to ensure that therapeutic failure does not occur during

pregnancy.

� 2009 Elsevier Ireland Ltd. All rights reserved.

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1. Introduction

An estimated 15–20% of women suffer from depression duringpregnancy necessitating treatment with antidepressants tomanage their symptoms [1]. Pregnancy-induced pharmacokineticchanges may affect the dose required to maintain effectivetherapy; these changes include expanded plasma volume, reducedplasma protein binding and increased cytochrome activity [2]. Ifthe need for dose increases during pregnancy goes unrecognizedthe depression may be sub-therapeutically managed leaving thewoman vulnerable to relapse.

* Corresponding author at: Division of Clinical Pharmacology and Toxicology,

Hospital for Sick Children, 555 University Ave., Toronto, ON, M5G 1X8, Canada.

Tel.: +1 416 813 7283; fax: +1 416 813 7562.

E-mail address: lobrien14@gmail.com (L. O’Brien).

0379-0738/$ – see front matter � 2009 Elsevier Ireland Ltd. All rights reserved.

doi:10.1016/j.forsciint.2009.12.034

Several studies have demonstrated pharmacokinetic changesduring pregnancy. The tricyclic antidepressants are one example ofmedications that have been shown to require greater doses inpregnancy to remain effective [3,4], due to their increasedmetabolism by the cytochrome 2D6 [5]. Conversely, less drugmay be required if enzymatic activity of metabolizing enzymes isdecreased, as is the case of CYP2C19 and the antimalarial drugproguanil [6,7].

However, when it comes to the SSRIs and SNRIs, there is apaucity of data regarding their pharmacokinetics in pregnancy.This lack of data is in part due to the invasive nature ofpharmacokinetic studies that would be required to collect serialblood samples over extended periods of time. One way toovercome these issues is to identify a biological matrix that canprovide the same information without the prolonged timecommitment or invasiveness of blood sampling.

Hair may serve as a non-invasive biological marker whichallows monitoring of long term systemic exposures to medications

Table 1Demographic data (n = 12).

Maternal characteristics

Age (mean� SD) 34.3�4.3

Gravidity (mean� SD) 2.5�1.3

Parity (mean� SD) 0.9�0.9

Race

Caucasian 83%

Hispanic 8%

Oriental-Asian 8%

Medication

Citalopram 33%

Fluoxetine 8%

Sertraline 8%

Venlafaxine 50%

L. O’Brien et al. / Forensic Science International 196 (2010) 93–9694

and other substances. As hair grows at a rate of approximately 1 cmper month [8], drug analysis in hair may allow for the identificationof metabolic activity throughout gestation. This is in contrast toblood which can only provide information regarding drugconcentrations at the time of sampling.

Our group recently described changes in the metabolism ofnicotine to cotinine during pregnancy using hair analysis. Thestudy was able to demonstrate that the ratio of hair nicotine:co-tinine decreased significantly between the first and thirdtrimesters of pregnancy, thus corroborating increased nicotinemetabolism in late pregnancy [9].

In the present study we compared for the first time,antidepressant metabolism in early and late pregnancy tometabolism in the postpartum period (>2 months after delivery)in a cohort of depressed pregnant women receiving SSRIs or SNRIs.Our primary objective was to determine whether the ratio of hairantidepressant:major metabolite changes in pregnancy.

2. Methods

The present study involved pregnant, depressed women consulting the

Motherisk program regarding the use of antidepressant medications during

pregnancy. The Motherisk program is a teratogen information service at the

Hospital for Sick Children in Toronto, Ontario which provides counseling on the

safety/risk of exposures during pregnancy and lactation. The protocol for this study

was approved by the Research Ethics Board of The Hospital for Sick Children in

Toronto (no. 000010045).

2.1. Subjects

Women taking antidepressant monotherapy for depression and who had no

other concurrent medical conditions or medication use at the time of their initial

call were included in the study (Table 1). Women were recruited in the first

Table 2GC–MS experimental conditions.

Substance Parent ion m/z [Da] Declustering pot

Norvenlafaxine 264 60

Venlafaxine 278 61

Sertraline 306 56

Fluoxetin 310 51

Desmethylcitalopram 311 55

Citalopram 325 81

Analytical conditions: The mobile phase consisted of (A) 0.1% (v/v) formic acid (Appl. Ch

solution, Fluka, Germany) and (B) methanol (gradient grade) containing 5 mM ammon

composition of 0% B, ramped to 100% B from 1 to 8 min with an isocratic post-run perio

temperature of 550 8C and gas flow settings (nitrogen as sprayer and heater gas) of 50

trimester of pregnancy (�12 weeks) and followed throughout pregnancy and into

the early postpartum period.

2.2. Hair sampling

At approximately 6 weeks postpartum women were contacted by telephone and

asked to provide a hair sample. A kit was subsequently mailed to each women that

included a detailed instruction booklet (with diagrams) describing how to cut the

hair, an information sheet asking about the date the hair was cut and any chemical

treatments the hair may have underwent and a sheet on which the hair was to be

affixed delineating the direction the hair should be placed in (i.e: scalp end and root

end). A postage paid return envelope was also included in the package.

Hair samples were obtained from the vertex posterior of the scalp and placed in a

clearly labeled envelope and stored at room temperature until ready for analysis.

2.3. Hair analysis

Hair samples were extracted and analyzed using liquid chromatography–mass

spectrometry–mass spectrometry (LC–MS–MS) using the extraction method and

conditions described by Thieme et al. [10] and Baumer [11] for amitryptiline

slightly modified for the SSRIs and SNRIs (Table 2).

Segmental analyses were performed on all hair samples. Each hair segment was

1 cm long, representing 1 month of hair growth, such that the segment most

proximal to the skull representing the most recent month. Each segment contained

at least 50 mcg of hair. The mean concentration of parent compound and metabolite

extracted from the hair was calculated for each trimester and the postpartum

period and reported in units of pg/uL, as the drugs were extracted from each

segment into a final volume of 0.3 mL. The limit of quantification for citalopram and

metabolite was 0.17 pg/mL while it was 0.33 pg/mL for fluoxetine, sertraline, and

venlafaxine and metabolites.

2.4. Correction for antidepressant metabolites

When calculating the antidepressant:major metabolite ratio for this study, a

correction had to be made to the metabolite concentrations in hair. The metabolites

of venlafaxine, citalopram, sertraline and fluoxetine are eliminated primarily by the

kidneys [12–14]. During pregnancy the glomerular filtration rate (GFR) increases

40–65% above non-pregnant levels [15]. This increase in renal clearance results in

the metabolites being cleared more quickly than in the non-pregnant state and

therefore blood and hair concentrations would reflect not only increased formation

rate but also increased elimination. To correct for this increased clearance of

metabolites, all first trimester and third trimester metabolite values reported were

corrected by 31% and 51% respectively [16,17]. A mean non-pregnant GFR baseline

of 96 mL/min was used.

2.5. Adherence

All women were asked to complete a ‘Missed Dose Diary’ for the duration of the

study period; women recorded whenever they missed a dose and how long they did

not take the medication for. Pharmacy records were also obtained to check

antidepressant prescription refill history.

2.6. Statistical analysis

Paired Student’s t-tests were used to compare the antidepressant:metabolite

ratios in the first trimester to the postpartum and the third trimester to the

postpartum. All statistical analyses were completed using the SPSS 15.0 for

ential [V] Product ion m/z [amu] Collision energy [eV]

246 19

58 35

260 17

58 37

275 15

159 39

148 13

117 55

293 23

262 23

109 37

262 29

em., Germany) with 5 mM ammonium formate (diluted from a 10 M aqueous stock

ium formate buffer and 0.01% (v/v) formic acid. A gradient program starting at a

d (8–8.5 min) provided sufficient separation at a flow rate of 700 mL/min. A source

psi were applied. The injection volume was 10 mL.

L. O’Brien et al. / Forensic Science International 196 (2010) 93–96 95

Windows software package. Statistical significance was defined as p < 0.05 for all

tests.

3. Results

Twelve women provided hair samples; 9 women provided hairthat was sufficiently long to encompass both the first and thirdtrimesters of pregnancy. No woman reported smoking or the use ofalcohol or any illicit drug during pregnancy (Table 1).

Citalopram doses ranged from 30 to 60 mg/day while venlafaxinedoses ranged between 75 and 300 mg/day. The dose of sertralinewas 75 mg/day while the dose of fluoxetine was 30 mg/day. Eightwomen remained on the same dose of medication throughout thepregnancy, while two increased their dose in the second trimesterand 1 woman decreased her dose continuously throughout thepregnancy. One woman stopped her medication altogether by thesecond trimester and the subsequent elimination of the medicationand its metabolite from the hair can be seen in Fig. 1.

The mean ratio of citalopram:norcitalopram in the first trimesterwas 0.89 � 0.26 versus 1.4� 0.24 in the postpartum period (p = 0.022).When the third trimester ratios were compared to those from thepostpartum period a statistically significant difference was also seen;0.9 � 0.14 and 1.4 � 0.24 respectively (p = 0.048). The mean ratio ofvenlafaxine and its metabolite was 1.1 � 0.4 and 1.03� 0.06 in the firsttrimester and postpartum period respectively (p = 0.774) while theratio in the third trimester was 0.80� 0.20 and was not statisticallysignificant when compared to the postpartum period (p = 0.192). Nostatistical analyses were conducted for sertraline or fluoxetine as theyeach had a sample size of one. The sertraline:norsertraline ratio was 5.8

Fig. 1. Hair profile of participant who discontinued antidepressant medication

(citalopram) after first trimester.

Fig. 2. Comparison of antidepressant:metabolite ratios in the first and third

trimesters.

in early pregnancy and decreased to 3.5 in late pregnancy. A largedifference was seen between the fluoxetine:norfluoxetine ratios in thefirst and third trimesters; 14 and 5.4 respectively. The mean ratio ofparent compound to metabolite concentration for each antidepressantis displayed in Fig. 2.

Adherence data was available for 6 out of the 9 women; theseparticipants filled their prescriptions on time and had an average of2.6 missed doses during the study period.

4. Discussion

This is a novel attempt to calculate antidepressant:metabolitemetabolic ratios in hair in demonstrating pregnancy-inducedchanges in drug metabolism. Our results indicate that citalopramshows a consistent increased metabolism in pregnancy whencompared to the postpartum period, while venlafaxine shows nosuch consistent difference in metabolism during these timeperiods, although there were documented individual cases withmarked changes. Single cases support pregnancy-induced meta-bolic changes for sertraline and fluoxetine. As shown in Fig. 1, thediscontinuation of the drug in one patient results in a steepdecrease in hair levels.

Our results for citalopram support the findings of Heikkinen etal. [18] who examined citalopram concentrations in blood duringlate pregnancy and compared them to levels in the postpartumperiod. Heikkinen et al. showed that metabolite:parent ratios weresignificantly higher in pregnancy than in the postpartum (orconversely parent:metabolite ratios were lower in pregnancy thanin the postpartum which is what our group found in this study). Arecent study by Sit et al. [19] corroborated increased metabolism ofcitalopram in late pregnancy when they examined the blood levelsof citalopram and its metabolite.

Venlafaxine is metabolized mainly by CYP2D6 which has beenshown to be increased in pregnancy [5]. Our results however, didnot demonstrate a persistent increase in metabolism duringgestation. To date there have been no systematic pharmacokineticstudies of venlafaxine in pregnancy.

When examining the older SSRI fluoxetine, one study [20]showed that fluoxetine metabolism increased in late pregnancythis is similar to the enhanced metabolism seen in the oneparticipant that took fluoxetine in our study.

This study was limited by its small sample size and the difficultyin obtaining hair samples of sufficient length to encompass anentire pregnancy. Further studies are needed to explore the utilityof this type of analysis for examining metabolic changes ofmedications in pregnancy.

5. Conclusion

Hair analysis offers a novel, non-invasive way to study themetabolism of antidepressant medications in pregnancy. It isimportant that variations in drug metabolism during pregnancy beconsidered as these changes may necessitate a dosage adjustmentto ensure that therapeutic failure does not occur during pregnancy.

Acknowledgements

Supported by a grant from the Canadian Institues for HealthResearch.

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