Drug Excretion into Breast Milk: Are all Drugs Contraindicated for Breastfeeding?
David A. Colantonio1, Sha Joshua Ye2, Sarah R. Delaney3, Ellie Majdi2,, Shinya Ito3
1Department of Pediatric Laboratory Medicine, Hospital for Sick Children, 555 University Ave. Toronto, ON, Canada, M5G 1Z82IONICS Mass Spectrometry, 32 Nixon Rd, Bolton, ON, Canada L7E 1W2
3Department of Pharmacology & Toxicology, University of Toronto, 1 King’s College Circle, Toronto, ON, Canada, M5S 1A8INTRODUCTIONRESULTS

Current epidemiological research provides strong evidence for healthbenefits associated with breastfeeding, including reductions in infantmortality, reduced chances of infection or the development of chronic
diseases, and positive impacts on cognitive development.1-3 Current
Centre for Disease Control Breastfeeding Report Card data
demonstrate more women (~ 80%) are breastfeeding and for longer.4
However, studies have also shown that 66 - 80% of women are onsome kind of medication during the postpartum period.5
Consequently, this leads to an increased likelihood of infant drug
exposure through breast milk. Although the dosages are considered
to be small, there have been reports of adverse events and evenfatality in infants exposed to drugs through breast milk. Although not
all drugs may be contraindicated while breastfeeding, there remains
little data on this topic.
Methotrexate (MTX) is the first line of treatment for rheumatoid
arthritis (RA), which has a high incidence in women of childbearing
age. Disease activity of RA often decreases during pregnancy but
tends to flare following delivery, necessitating the reestablishment oftreatment. Only one case report on the excretion of MTX into breast
milk has been published. Due to the paucity of data on MTX levels inbreast milk, the potential risk of toxicity and drug accumulation in theinfant remains largely unknown. We have developed a sensitive andspecific LC-MS/MS method to quantitate MTX and its metabolite inhuman milk and applied it to patient samples. We have also
calculated the relative infant dose of MTX to determine the risk to theinfant. The objective of this study is to investigate the risk of drug
exposure in nursing infants by determining drug concentrations inbreast milk, with an aim to developing pharmacokinetic profiles fordrugs excreted into breast milk.

METHOD
Sample Preparation
To investigate infant drug exposure through breast milk, weestablished a drug safety monitoring program, Drugs in Lactation
Analysis Consortium (DLAC), to measure several drugs commonlyused by women breastfeeding, beginning with methotrexate. Breastmilk is a complex lipid and protein-rich matrix, with drugs partitioning
to either the aqueous or lipid phase, thus requiring meticulous
sample processing before analysis. Differences in the lipid andprotein composition of milk exists between fore and hind milk. We
first investigated whether drugs partition to the aqueous or the lipid
phase of breast milk. We then worked to create a simplified drug
extraction method using hexane, methanol and acetonitrile tofacilitate efficient drug extraction from breast milk, as shown below.

Calibration Curves

Pharmacokinetic profilinga) Methotrexateb) 7OH-Methotrexate

0.7525ng/mL
R2=0.9999

0.7525ng/mL
R2=0.9997

Fig.1ab. Calibration curves for (a) Methotrexate and (b) 7OH-Methotrexate in breast
milk .

Good linearity (with R2 > 0.999) was obtained for methotrexate andits metabolite 7OH-methotrexate. Multiple replicates (8 in severalruns) were performed. Examples of the calibration curves generated
for methotrexate and 7OH-methotrexate are shown in Figures 1ab,respectively. Excellent linearity was obtained for a concentration
range of 0.75 to 25ng/mL for methotrexate and 7OH-methotrexate.
The accuracy obtained is between 90.0% to 106.3% and thecorresponding CV% is less than 11.3% within a run.
A between day precision study was also carried out using 3 levels ofa QC sample injected multiple times over multiple days. The mean,standard deviation and CV were determined. The results in Table 2(below) show that for QC levels 2 & 3, the CV% is less than 6%.
Mean(ng/mL)

Stdv% CVQC Level 1

1.21

0.27

22.3

5.3

QC Level 2

8.20

0.37

4.5

2.0

QC Level 3

17.62

0.50

2.8Methotrexate

Mean(ng/mL)

Stdv% CV7-OH Methotrexate

QC Level 11.03

0.24

23.6

QC Level 2

7.49

0.40

QC Level 3

16.77

0.33

Table 2ab: Mean, standard deviation, and CV for three levels of QC for methotrexate
and 7OH-methotrexate.

Method Comparison

Column:Imtakt Unison
UK-C8, 75x3mm, 3µm
Flow Rate:
0.6 mL/min
Injection Volume:
10 µL
Column Temperature: 30CTime (min)Solvent B %

0.3

20

1.895

2.095

2.1

103.5

10Table1: LC gradient, 3.5 min
total runtime

www.ionics.ca

Pharmacokinetic profiling of methotrexate in breast milk following a
subcutaneous dose of 25 mg/mL was examined. Both foremilk andhindmilk were analyzed. As illustrated in the Figure 3, methotrexate
concentration in breast milk peaks between 1 – 12 hours post doseand quickly decreases within 24 hours post-dose. By 48 hours postdose, the concentrations excreted in breast milk are negligible.
Interestingly, methotrexate is excreted into breast milk with nonotable differences in drug concentrations between foremilk andhindmilk. These data provide the foundation to establish a TDM
system for measuring drug concentrations in breast milk. We hope
to carry out population-based pharmacokinetic analysis to establish
safety guidelines on drug excretion into breast milk.

CONCLUSION
Good linearity and reproducibility were observed for methotrexate
and its metabolite 7OH-methotrexate in both serum and breast milk.
The results indicate that this ESI-LC-MS/MS method using anIONICS 3Q 220 mass spectrometer providing the best ionization andsampling efficiencies is robust and ideal for this clinical researchstudy.
This study highlights the issue of infant risk for toxicity though drug
exposure in breast milk. Methotrexate is excreted into breast milk at
significant concentrations within the first 24 hours post-dose.
However, no notable differences in drug concentrations between
foremilk and hindmilk were observed. Due to the difficulty inobtaining foremilk and hindmilk, this is the first study to measure andcompare drug levels in this sample type. These data provide thefoundation to establish a TDM system for measuring drugconcentrations in breast milk, with the aim to carry out populationbased pharmacokinetic analysis to establish safety guidelinesregarding drug excretion into breast milk as well as breast feeding
guidelines.

Then 5µL of supernatant were loaded on an analytical column andeluted with an LC cycle time of 3.5 minutes. The signal is detected by
an IONICS 3Q 220 triple quadrupole mass spectrometer. All solventsare HPLC grade.
Instruments
A Shimadzu Prominence UFLCxr system was coupled to the IONICS
3Q 220 mass spectrometer. The detailed LC conditions are shown
below and the time program is listed in Table 1.

Fig.3. Pharmacokinetic profiling of methotrexate in breast milk following a
subcutaneous dose of 25 mg/mL. Both foremilk and hindmilk were measured.

References:Fig.2. Method comparison between one sample preparation for serum and themethod developed for breast milk in this study.

Fourteen serum samples from patients on methotrexate, who had drugconcentrations determined by another LC-MS method, were also
analyzed using the method developed for this study. The plot in Figure 2
shows good agreement between methods with an R2-value of > 0.95
and a percent difference between results of < 20%.

1¸ Section on breastfeeding, American Academy of Pediatrics: Policy Statement.
Breastfeeding and the use of human milk. Pediatrics 2012; 129: e827-841.
2. Cunningham AS, Jelliffe DB, Jelliffe EFP, Breast-feeding and health in the1980s: A global epidemiological review. J Pediatrics 1991; 118: 659-66.
3. Ruiz-Palacios GM, Calva JJ, Pickering LK, Lopez-Vidal Y, Volkow P, Pezzarossi
H, West MS. Protection of breast-fed infants against Campylobacter diarrhea byantibodies in human milk. J Pediatr 1990; 11: 707-13
4. Centre for Disease Control Breastfeeding Report Card USA 2014.
5. Schirm E, Schwagermann MP, Tobi H, de Jong van den Berg LTW. Drug use
during breastfeeding. A survey from the Netherlands. Eur J Clin Nutr 2004; 58:386-390.

*For Research Use Only