Study setting and design

This study was conducted at The King’s Daughters Milk Bank (KDMB), one of 32 not-for-profit donor human milk banks in North America accredited by the HMBANA. KDMB collects, processes, and distributes PDHM to medically fragile infants in NICUs, newborn nurseries, and outpatients with medical need on the Eastern seaboard. During processing of 977 batches, from December 2022 to June 2024, a total of 150 random samples of pooled PDHM were collected for drug analyte testing. Pooled samples were aggregated from an average of 5 donors (range of 4–8), with a total of 742 donor contributions in the 150 tested samples. Informed consent was obtained from all donors as part of the standard intake process. This study was approved by the Institutional Review Board and Human Subjects’ Protections at Macon & Joan Brock Virginia Health Sciences at Old Dominion University (IRB#: 22-11-FB-0225).

Donor screening procedures

Per HMBANA Standards, the KDMB employs a comprehensive three-step screening process for all potential donors:

Phone interview

Applicants participate in a 20 min telephone interview with a trained staff member, covering topics such as current infant feeding practices, surplus milk availability, milk collection and storage details, and the health history of both the mother and infant. The KDMB interview includes specific drug screening questions such as “In the past 12 months, have you used any street drugs or illicit drugs, including marijuana?” and “In the past 12 months, have you used any prescription medication just for the feeling, more than prescribed, or that were not prescribed for you?” Affirmative answers to these questions require further investigation and may result in a period of milk deferral or disqualification as a donor per HMBANA Standards [12].

Electronic questionnaire and healthcare provider communication

Applicants passing verbal phone screening then complete an electronic questionnaire with additional drug screening questions, including “In the past 5 years, have you used recreational drugs such as marijuana, cocaine, LSD, ecstasy, amphetamines, or prescription medications not prescribed for you?” and “In the past 12 months, have you ingested, smoked, or applied CBD or THC products?” Affirmative answers to these questions require further investigation and may result in a period of milk deferral or disqualification as a donor per HMBANA Standards [12]. Additional items also inquire about high-risk behaviors that extend beyond the individual donor to include their household members and sexual partners. KDMB also communicates with both maternal and infant healthcare providers to gather information about maternal health, prenatal serologies, medications, history of substance abuse, and infant health concerns.

Serologic testing

Applicants undergo serologic testing for hepatitis B, hepatitis C, HIV, human T-lymphotropic virus, and syphilis. Donors with positive tests are deferred or disqualified per HMBANA Standards [12].

Donor approval is granted only after review of all application documentation by the KDMB milk bank director (BSN, RN, IBCLC) and medical director (MD, IBCLC).

Milk collection and processing

Breast milk is expressed by donors in their personal environment using either manual or electric pumps. The milk is stored in dated human milk storage bags and promptly refrigerated and frozen within 96 h of expression. Frozen milk is delivered or shipped overnight to the milk bank. For batch processing, milk from 4–8+ approved donors is selected, thawed, pooled, bottled, and labeled. The batches are then pasteurized using the Holder Method (62.5 °C for 30 min) and immediately refrozen. One bottle from each batch is chosen at random and sent to a Clinical Laboratory Improvement Amendments (CLIA)-accredited lab for microbiological culture; any batch testing positive for microbial growth is discarded.

Sample collection for drug testing

During processing, one bottle of batched milk contains the temperature-sensing probe. This bottle is typically discarded after processing. For this study, a small aliquot of milk was collected from this discard bottle for drug testing. Samples were collected from each batch of pooled, PDHM processed during the study period. The random frozen samples were shipped on dry ice to the InfantRisk Center of Excellence Laboratory at Texas Tech University Health Sciences Center for testing.

Analytical testing methods

The selection of drug analytes for testing was based on substances with the highest prevalence of use in the local population, as determined through consultations with the state drug laboratory and review of national drug use publications. This targeted approach focused on amphetamines (methamphetamine, MDMA), benzodiazepines (alprazolam), opioids (fentanyl, hydrocodone, hydromorphone, 6-acetyl morphine, morphine, oxycodone, oxymorphone), cocaine and its metabolite benzoylecgonine, and marijuana compounds (Delta 9-THC, 11-OH THC, 11-COOH THC), representing the most common substances of concern for potential exposure in this population. The analytes were obtained from Cerilliant corporation, division of Sigma–Aldrich. Deuterated internal standards for these analytes were also sourced from Cerilliant Corporation. All the standard solutions received were already prepared in solution and were further diluted with methyl alcohol for subsequent method development and analysis.

LC-MS/MS analyses were performed using a Shimadzu LC-40 system connected to an AB Sciex 7500 mass spectrometer system in Multiple Reaction Monitoring (MRM) mode, operating the source in positive ion mode, equipped with an electrospray (ESI) ion source. Table 1 describes the 15 analytes, which were tested, with their respective limits of quantification (LOQ). Chromatographic separation was achieved at 40 °C using a Restek Biphenyl column (100 mm × 2.1 mm i.d., 2.7 μm particle size). The mobile phase consists of solvent A: (2 mM ammonium formate and 0.2% formic acid) and solvent B: (methyl alcohol: Acetonitrile 70:30 v/v, 0.2% formic acid and 2 mM ammonium formate). The flow rate was kept constant at 0.4 mL/min during the analysis, and the sample volume injected was 5 μL, and followed by gradient elution. Acquisition was performed in multiple reaction monitoring (MRM) mode. For sample extraction, 100 μL of milk sample (including blanks, calibrators, QC, and real samples), 10 μL of deuterated internal standard mix (5 ng of each analyte), and 400 μL of acetonitrile were added. The mixture in the tubes was vortexed for 5 min and centrifuged at 14,000 rpm for 10 min at 4 °C temperature. The supernatant was transferred, the organic layer evaporated, and reconstituted in 100 μL of 10% of solvent A as described above. For cannabinoids extraction, 100 μL of human milk was used along with 10 μL of mix of a deuterated internal standard mix (100 ng stock). After adding 300 μL of acetonitrile, mixture was vortexed, centrifuged, supernatant was dried. The final sample was reconstituted in 100 μL of water: methyl alcohol (15:85 v/v).

Table 1 Compounds analyzed and their limits of quantification (LOQ).

Method validation included linearity, limits of detection (LOD) and quantification (LOQ), imprecision, accuracy, selectivity, matrix effect, and recovery. Linearity was determined by least-squares regression with 1/x2 weighting. Calibration curves in blank human milk were validated across a range of 0.039–5 ng/mL for all analytes and their limit of quantification described in Table 2. Linearity was considered acceptable with the coefficient of determination (R2) of at least 0.99. Quality control standards were prepared at low, medium, and high concentrations for all analytes.

Table 2 Oxycodone quantification and interpretation in the PDHM samples >LOQ.