No time to cry: the detectives analyze infant formula

Chapter 17
📁 Case overview: The detectives are so impressed by the cuteness of a newborn, that they immediately take on a case on quality and safety assurance in infant formula. Will their analysis of harmful compounds, 3-MCPD and glycidol, in infant formula win them a giggle or a gush of tears? Read on to find out.

Eggcule Poirot is very excited. His sister, P-egg-y has been quite pr-egg-y for some time and has just given birth to a beautiful baby girl. The whole team tags along as they go visit the cute little baby. After they are finished cooing and wowing at how adorable she is, they slowly head back to the office and back to work.

Shallot Holmes digs through the cases that are waiting for their attention and chuckles happily as he pulls one file out. This client is interested in achieving safety assurance of infant formula. The others look at each other and smile happily at the thought of continuing the baby connection for a little longer.

As he has just become an uncle, Eggcule Poirot inherits the honour of leading the case. The detectives sit themselves down comfortably and look up at him as he paces slowly, scratching his chin as he collects his thoughts. He faces them and gives them an overview of quality and safety assurance of infant formula. He does acknowledge he is now heading a completely different liquid than in his last case, where he led the team into successful alcohol determination in wine.

He begins by narrowing in on one of the substances that needs careful monitoring in infant formula, 2-and 3-MCPD (monochloropropanediol). Esters of 2-and 3-MCPD are formed during the refining process of vegetable oils. The formation of 2-and 3-MCPD from glycerol in presence of chlorideions takes place at high temperatures during the deodorization step, followed by esterification with free fatty acids. Therefore, any food that contains vegetable oils, including infant formula, can contain 2- or 3-MCPD esters.

How much of the substance is present in the food depends on the type of oil used. Palm oil, for example, has the highest content of 3-MCPD. Free 3-MCPD can also form during the heating process, such as grilling of meat, and during the hydrolysis of vegetable proteins that are used in food items such as soy sauce.

Another substance that Eggcule Poirot draws the team’s attention to is glycidyl estersin. The only known source of this compound in food is refined vegetable oil. There, glycidyl estersin appears to form during the heating of diacyl glycerol or monoacylglycerol under the high temperature conditions of deodorization.

With the chemistry background out of the way, Eggcule Poirot explains why these substances are of importance.

3-MCPD has been classified as potentially carcinogenic to humans and glycidol as carcinogenic and genotoxic.

The detective briefly mentions that the glycidyl esters themselves are not toxic, but they are released in human digestion and that both free and bound 3-MCPD is taken into account to estimate food safety levels.

Eggcule Poirot performs a quick search on his laptop, connects the device to their white wall and shows them a few important facts. A tolerable daily intake (TDI) for 3-MCPD of 2 μg/kg body weight is accepted by the Joint FAO/WHO Expert committee on Food Additives – JECFA and the German Federal Institute for Risk Assessment (BfR), whereas the European Food Safety Authority (EFSA) has defined a lower TDI value of only 0.8 μg/kg body weight. There is no TDI value established for glycidol and glycidylesters, but their presence should be avoided according to the As Low As Reasonable Achievable (ALARA) principle.

Miss Mapple asks about the relevance of these substances to infant formula. Eggcule Poirot is, for once, happy with the interruption, as he was just about head into this direction himself.

The detective points out that among all foods in danger of 3-MCPD or glycidol contamination, infant formula is the most critical one because baby formula is the sole food that is consumed by babies that are not breasted and their body weight is very low.

Lieutenant Cornlumbo raises his hand to ask how these substances are analyzed.

Eggcule Poirot is once again pleased with the question and already prepared with an answer. 3-MCPD should not be extracted using methods with acidic or alkaline hydrolysis, such as Weibull-Stoldt or Mojonnier, because significant amounts of 3-MCPD esters form during these procedures. Instead, pressurized solvent extraction can be used to determine the levels of 3-MCPD in infant formula and other food items, such as grilled meat and meat products.

With this in mind, Eggcule Poirot proposes that they provide their client with a BfR pressurized solvent extraction method for analysis of 3-MCPD in infant formula. Naturally, the detectives have to test out the method first and they spring into action.

First order of business is gathering samples and appropriate standards. After a quick shopping spree in the local testing laboratory and grocery store, they come and back and lay out their inventory. It consists of:

Standard solutions:

– Solution 1: 20.1 mg rac 1.2 Bis-palmitoyl-3-chloropropanediol diluted in 100 mL ethanol (0.201 mg/mL); 1 mL diluted to 10 mL in MTBE (0.0201 mg/mL). 18.48% of rac 1.2 Bis-palmitoyl-3-chloropropanediol is equal to 3-MCPD, therefore standard solution 1 contains 0.003714 mg/ml 3-MCPD

– Solution 2: 22.6 mg glycidyl stearate diluted in 100 mL ethanol (0.226 mg/mL); 1 mL was diluted to 10 mL in MTBE (0.0226 mg/mL). 21.67% of glycidyl stearate is equal to glycidol, therefore standard solution 2 contains 0.004897 mg/mL glycidol.

Infant formula samples:

– Infant Formula A: PRE, from birth, expected fat content: 25.20%

– Infant Formula B: stage 2, after 6 months, expected fat content: 21.50%

Since there are no certified reference materials available for the determination of 3-MCPD and glycidol in infant formula, the detectives decide to spike the samples with 3-MCPD and glycidol.

The private investigators proceed to put their heads together to come up with a plan of action. Eggcule Poirot jots down the main steps of their procedure as follows:

1. Extract infant formula using a pressurized solvent extraction system
2. Evaporate extract to dryness for fat determination using a parallel evaporation system
3. Determine 3-MCPD and glycidol in the extracted fat using DGF method C-VI

They quickly start with the first step with the following detailed method:

– They weigh 2 g of the sample and 4.6 g DE into a beaker
– They add 0.8 mL water and mix well with spatula
– The load the samples into the cells of the instrument using a funnel.
– They spike the samples by adding standard solutions into the samples using an Eppendorf pipette:

• 0.203 mL of standard solution 1 (corresponding to 0.00075 mg 3-MCPD)
• 0.152 mL of standard solution 3 (corresponds to 0.00075 mg glycidol).

– They dry 240 mL collection bottles in a drying oven at 102°C for 30 min.
– They cool down the collection bottles to ambient temperature in a desiccator for 60 minutes.
– They weigh and record the bottle weights, with accuracy of 0.1 mg.
– They place the collection bottles in the receiving unit and carry out the extraction using the following parameters:

After the extraction, the detectives proceed to step 2 of their overall process. They evaporate the samples using the parameters as listed below:

The team then goes on to:

– Dry the collection bottles in a drying oven for 30 min at 102 °C.
– Cool down the bottles in a desiccator for 60 minutes.
– Weight the bottles containing the extracting the record the weight.
– Calculate the fat content using the following equation:

Where:

%Fat = percentage of fat in the sample
m_total = vial weight + extract (g)
m_vial = vial weight (g)
m_Sample = sample weight (g)

The detectives are now finished with two out of three steps. They look at each other satisfied, take a brief break and go on to the final step: determination of 3-MCPD and glycidol in the extracted fat of the infant formula samples.

Eggcule Poirot gives them a quick rundown on the theory. They will perform the determination according to the DGF C-VI 18 standard method. This procedure involves the determination of free and bound 3-MCPD after alkaline catalyzed ester cleavage and derivatization with phenyboronic acid (PBA). The method also allows the indirect determination of glycidyl esters under the assumption that no other substances are present that can react at room temperature with inorganic chloride to generate 3-MCPD. The quantification of the derived compounds is accomplished through GC-MS, using deuterated surrogate standards. The method is based on the following principle:

Assay A: Determination of the sum of bound 3-MCPD and bound glycidol
Assay B: Determination of the bound 3-MCPD.

The total amount of bound glycidol is then calculated as the difference between these two assays.
With this knowledge in their back pocket, the detectives are eager to get their hands dirty. The continue their assessment by following these steps:

1. They take an aliquot of 100 mg of extracted fat for each assay.
2. They weigh this aliquot into a 1.5 ml screwcap vial.
3. The add 100 µL of the surrogate standard and 100 µL of MTBE to each assay and shake it until the sample is completely dissolved.
4. They add 200 uL of sodium hydroxide to each assay and shake it for a short time.
5. They wait briefly. The reaction time for the ester cleavage is between 3.5 to 5.5 min.
6. They stop the ester cleavage by adding:
– 600 μL of acidified sodium chloride solution to assay A
– 600 μL acidified sodium chloride solution to assay B
7. They add 600 µL of isohexane, shake the vials and wait for 5 minutes.
8. They shake both assays and separate and discard the organic phase, then they repeat steps 6 and 7 once more.
9. They extract the aqueous phase of both assays three times with 600 µL of a mixture of diethyl ether (ethyl acetate).
10. They combine the organic phases in a vial containing sodium sulphate.
11. The add the derivatization reagent (10 – 100 mL, depending on the capability of the chromatography system)
12. The evaporate both assays to dryness using a gentle nitrogen stream.
13. The redissolve the dried extracts with 500 µL of isooctane.
14. They perform GC-MS according to the following conditions:

They breathe out a long sigh of relief once all the steps for 3-MCPD and glycidol determination in infant formula have been completed.

They then collect their data and get set for some serious calculations needed for safety assurance of infant formula.

First they review their spiked amounts.

Solution 1: 20.1 mg rac 1.2-Bis-palmitoyl-3-chloropropanediol was diluted in 100 mL ethanol (0.201 mg/mL); 1 mL was diluted to 10 mL in MTBE (0.0201 mg/mL). 18.48 % of rac 1.2-Bis-palmitoyl-3-chloropropanediolis equal to 3-MCPD, therefore standard solution 1 contains 0.003714 mg/mL 3-MCPD. 0.203 mL of standard solution1 was added.

Therefore mspike= 0.00075404 mg 3-MCPD.

Solution 2: 22.6 mg Glycidyl stearate was diluted in 100 mL ethanol (0.226 mg/mL); 1 mL was diluted to 10 mL in MTBE (0.0226 mg/mL). 21.67 % of glycidyl stearateis equal to glycidol, therefore standard solution 2 contains 0.004897 mg/mL glycidol. 0.153 mL of standard solution 2 was added.

Therefore mspike= 0.00074931 mg glycidol.

The detectives nod satisfied and continue to calculate the recovery for spiked samples:

Recovery is calculated as a relation of measured analyte to total analyte (spike amount + measured analyte):

Where:
m_{fat, cal}: : calculated fat amount [g]
m_sample: used sample amount [g]
%Fat: measured fat content [%]
m_spike, cal: relation of spike to fat [mg/kg]
m_spike: amount of added spike [mg]
m_analyte, total: total amount of analyte: 3-MCPD resp.glycidol[mg/kg]
m_analyte: measured amount of 3-MCPD resp. glycidol in non spiked sample [mg/kg fat]
Recovery: relation of measured analyte to calculated analyte[%]
m_analyte, spike: measured amount of 3-MCPD resp. glycidol in spiked sample [mg/kg fat]

After double-checking their calculation, the detectives examine their results.

Fat determination in infant formula A; expected fat content: 25.2%:

Fat determination in infant formula B; expected fat content:21.50%

Eggcule Poirot examines the results carefully and begins to analyze them. The determined fat content of sample A shows a good recovery compared to the declared fat content. For Sample B, the determined fat content is lower as expected. He shows a paper he pas pulled showing determined fat contents in infant formula using pressurized solvent extraction are typically in a recovery range of 78.5 to 101.8 % (Wöhrlin et al.). In their experiments, their samples are only infant formula of stage PRE and 1. Due to different composition of infant formula of different stages, the recoveries for fat determination vary. The extraction method they have used is optimized for determination of 3-MCPD and glycidol, not for fat determination. The determination of fat content is only necessary for the calculation and interpretation of the spiked samples. Therefore, the fat determination of sample B is acceptable.

However, not to be outdone as a master in fat determination as well, Eggcule Poirot offers his colleagues a nice application note on fat determination in infant formula in case they want to learn how to optimize the process indeed.

The detectives then analyze their “native” samples or those of infant formulas without addition of spiking solutions.

Measured amount of 3-MCPD and glycidol in fat, not spiked sample (infant formula A):

Measured amount of 3-MCPD and glycidol in fat, not spiked sample (infant formula B):

Eggcule Poirot compares their data to those of recent publications showing that in infant formula, the 3-MCPD content is 0.25 to 0.65 mg/kg extracted fat resp. the glycidol content is 0.16 to 0.81 mg/kg extracted fat (Wöhrlin et al.). The detective is happy to note that the 3-MCPD and glycidol content in infant formula in their assays are comparable to these results.

They move on to the spiked samples where the addition of spiked solution is equivalent to 1.5 mg/kg -2 mg/kg fat.

Measured amount of 3-MCPD and glycidol in fat in spiked sample (infant formula A):

Measured amount of 3-MCPD and glycidol in fat in spiked sample (infant formula B):

Their data for recovery of spiked samples looks as follows:

Recovery of 3-MCPD spiked in infant formula A (measured fat content: 25.00%)

Recovery of 3-MCPD spiked in infant formula B (measured fat content: 18.87%)

Recovery of glycidol spiked in infant formula A (measured fat content: 25.00%)

Recovery of glycidol spiked in infant formula B (measured fat content: 18.87%)

The detectives examine their results. Eggcule Poirot begins to think out loud. From literature, expected recoveries for the 3-MCPD are in the range of 92 to 109% (Wöhrlin et al.). The results for infant formula A correspond to this range. The recoveries for infant formula B are higher than published data. Eggcule Poirot reasons that his could be due to the lower recovery of fat extraction (as shown in the previous tables). Assuming that the spiked stearates are extracted easier than the native fat of the sample, this results in an overestimation of the 3-MCPD and the glycidol per gram of fat. The detective offers the possible solution to correct the recovery by this factor if the fat recovery is too low. If the determined amount of 3-MCPD and the glycidol is corrected by the fat recovery, the recovery is around 100 to 100%, which is comparable to results in literature as mentioned above. Infant Formula A, on the other hand, does not require any corrections, since fat determination was at 100% recovery. No correction is needed for native samples, since the 3-MCPD is bound to the native fat matrix.

Shallot Holmes takes a minute to process all the information fed to them by Eggcule Poirot. He nods his head in agreement, as do the others.

Corrected recovery of spiked 3-MCPD and glycidol in infant formula B (measured fat content: 18.87%, fat recovery 88.7%)

There is one last thing left for the detectives to deal with. They must compare their method to standard methods. Eggcule Poirot finds data from an alternative pressurized solvent extraction method published by the ESFA. The detectives summarize and compare their parameters with that of the ESFA method in a table:

The detectives discuss how they can present their findings to the customer.

Eggcule Poirot summarizes what their data reveals about infant formula. The sample infant formula A is meant for use directly after birth. It is recommended to feed a baby of this age with 100 mL liquid infant formula, six times per day. For 100 mL liquid infant formula, 13.5 g powder is required for a total of 81 g per day. 81 g of powder contains 20.25 g of fat (fat content measured: 25%). The measurements of the detectives have shown a 3-MCPD content of 0.52 mg/kg fat resp. glycidol content of 0.105 mg/kg fat in infant formula A. This means per day, 0.01052 mg 3-MCPD and 0.0213 mg glycidol will be consumed by the baby. Taking the average body weight of 3.5 kg, the baby’s TDI would be 3.01 µg/kg of 3-MCPD, which Is by a factor of 3.8 higher than the TDI of 0.8 µg/kg body weight (TDI published by EFSA). Glycidol should also be avoided, but the daily intake is 0.61 µg/kg body weight.

Infant formula B is recommended for children over 6 months. At this age, babies drink about 600 mL of infant formula per day. For the preparation of 600 mL of infant milk, it is necessary to use 78 g powder of infant formula B. 78 g powder contain 14.72 g of fat (fat content measured: 18.87 %). The measurements of the detectives show a 3-MCPD content of 0.36 mg/kg fat resp. a glycidol content of 0.32 mg/kg fat in infant formula B. This translated to daily levels of 0.0053 mg 3-MCPD and 0.0047 mg glycidol that are consumed by the baby. Assuming a body weight of 7.5 kg, the daily intake would be 0.71 μg/kg of 3-MCPD which is below the TDI of 0.8 μg/kg body weight. For glycidol, the daily intake is 0.63μg/kg.

Satisfied with their analysis, the detectives hand over their work to Eggcule Poirot who heads out to present their findings to the client. He returns satisfied as the customer is pleased with their procedure for the extraction of infant formula using pressurized solvent extraction , as a fast and reliable method for determination of 3-MCPD and glycidol levels.

Incredibly exhausted, the detectives pass on the opportunity for a celebration and head on home. Each of the detectives collapses in bed to enjoy a true gift that those around a newborn would most appreciate, a full night of uninterrupted sleep.