Skin penetration of Vitamin C (Ascorbic acid): Part II

“Applying 15% Vitamin C for three consecutive days creates a reservoir effect in the skin.”

Firstly, I want to remind you that this study was done on pig skin – not humans. The way that ascorbic acid is stored and metabolized in pig skin may vary from human skin.

Most animals, like pigs, are able to synthesize their own ascorbic acid from glucose, but humans cannot. It’s possible that this data from pigs will be similar to human data, but it’s also very possible that it won’t be. Neither has been proven yet. Presenting an assumption as truth is misleading – but often done in marketing.

I also want to remind you that the way that the ascorbic acid was applied to the skin was not the same way that we apply our skincare. In these experiments, the ascorbic acid solution was applied with a Hill Top Chamber, which occludes the skin, reducing evaporation and theoretically enhancing skin penetration.

For this part of the experiment, Pinnell and his group applied a 15% ascorbic acid solution at pH 3.2 to pig skin for 5 days with a Hill Top Chamber. After the 5th day, application of the ascorbic acid was stopped and ascorbic acid levels in the skin were monitored for an additional 5 days.

After the 3rd day of application of the ascorbic acid serum, the ascorbic acid levels in the skin do appear to reach a peak around 1100 pmol/mg. The deviation around the mean does appear to be reducing with each further day between the 3 subject pigs.

We do need to consider if this theoretical peak amount of ascorbic acid is reached in real-life situations. The living conditions of the pigs in the study were not described, so it’s possible that they were not exposed to natural daylight. It’s understood that UV exposure reduces the amount of ascorbic acid in the skin. UV increases the production of free radicals in the skin, and ascorbic acid is part of the natural antioxidants in the skin which help neutralize these free radicals.

In an experiment using human skin models, it was found that exposure to 16.9 joules/cm² (About 12 minimal erythemal dose equivalent) of UV reduced ascorbic acid levels in the skin model by almost ⅓. This was a higher amount of UV exposure the experimenters expected, they were also unable to detect dehydroascorbic acid in the skin. The study does have some issues which “may be explained by the high levels of ascorbate present in the [tissue] medium…added by the manufacturer to increase collagen synthesis”.

“Vitamin C remains in the skin for 3-4 days and doesn’t wash out”

This marketing claim may be due to some confusion of the term “washout”. In drug experiments a “washout period” refers to the period of time when treatment is stopped, it does not necessarily mean that the skin is washed out.

After applying the 15% ascorbic acid solution to the pig skin, they discontinued application and monitored ascorbic acid levels in the skin. Unfortunately, the methodology in this portion of the experiment isn’t explicitly described. It is unclear, for example, if the pig’s skin was washed each day. The washing procedure is described as “…at the end of the experiment, the formulation was washed vigorously from the skin with water.”

Because most of us use surfactant based cleansers to wash our skin, this data may not be as applicable as the pig’s skin was washed with only water. However, the pig’s skin was removed of stratum corneum before ascorbic acid measurement and the lower layers of skin are likely less affected by the washing and surfactant based-cleanser.

Based on this data, the half-life (the amount of time it takes for the detected ascorbic acid levels to drop by half) was estimated at around 4 days. But as mentioned above, it’s unclear what the living conditions of the pigs were and whether or not they were exposed to sunlight which reduces antioxidant levels in the skin.

Can Vitamin C derivatives increase levels of Vitamin C in skin?

The last portion of the Pinnell experiment looked at whether or not the topical application of Vitamin C derivatives could increase levels of Vitamin C as ascorbic acid in pig skin.

For 24 hours, solutions of dehydroascorbic acid, 10% ascorbyl-6-palmitate, 12% magnesium ascorbyl phosphate, and 15% ascorbic acid were applied to pig skin. Compared to control, only the 15% ascorbic acid solution created a statistically significant increase in ascorbic acid levels in the skin.

For the derivatives, there was no statistically significant difference between the application of the derivative and control (no application of derivatives or Vitamin C) – which implicates that, at least for pig skin, these specific derivatives do not convert to Vitamin C.

For the solutions of dehydroascorbic acid, pig skin levels of ascorbic acid were 7.51 ± 3.34 pmol/mg for 20 mM dehydroascorbic acid and 8.70 ± 2.13 pmol/mg for 1 M dehydroascorbic acid. Where no dehydroascorbic acid was applied levels of ascorbic acid were 9.24 ± 3.55 pmol/mg.

In conclusion…

It surprises me how influential this one study on ascorbic acid applied to pig skin has become in terms of marketing language for brands.

Even later studies with Dr. Pinnell as an author leave out that the data are collected from pig skin, “Following topical application, once the skin is saturated with L-ascorbic acid, it remains with a half-life of about 4 d (Pinnell et al, 2001).”

While this experiment is some of the best data we have in terms of ascorbic acid penetration based on formulation, the key point to remember is that human skin cannot be assumed to behave the same as pig skin.

So if you see a claim similar to “15% Vitamin C at pH 3.5 is the most effective concentration”, please imagine me beside you whispering “…for pigs”.

Edit: An error was made in the original version published, pigs can synthesize Vitamin C from glucose, but humans can not. Guinea pigs also cannot synthesize their own Vitamin C.

Edit: An error was made in the original version published, pmmol was corrected to pmol.

Source: Pinnell, S. R., Yang, H. , Omar, M. , Riviere, N. M., DeBuys, H. V., Walker, L. C., Wang, Y. and Levine, M. (2001), Topical L‐Ascorbic Acid: Percutaneous Absorption Studies. Dermatologic Surgery, 27: 137-142. DOI: 10.1046/j.1524-4725.2001.00264.x

Podda, M., Traber, M.G., Weber, C., Yan, L., Packer, L. (1998), UV-Irradiation Depletes Antioxidants and Causes Oxidative Damage in a Model of Human Skin, Free Radical Biology and Medicine, 24: 55-65. DOI: 10.1016/S0891-5849(97)00142-1

Skin penetration of Vitamin C (Ascorbic acid): Part I

Today I wanted to look at a research paper primarily led by Dr. Sheldon R. Pinnell. He is one of the founders of Skinceuticals and contributed much of the early research on the use of Vitamin C as ascorbic acid on skin. He and his group also discovered the synergistic effect of Vitamin C, Vitamin E, and Ferulic acid – which is commonly used in many products on the market today.

The data from this paper is often quoted in marketing material for Vitamin C serums, but one extremely important piece of information is often left out – the data was collected from pigs, white Yorkshire pigs to be exact.

Many people also have ethical concerns when it comes to the use of animals in cosmetic research. Synthetic and lab grown human skin equivalents are being researched and tested which will one day replace the use of animal as well as human testing in cosmetics.

It should be clear that human skin and pig skin are not the same, but they do have similar properties which is why it is often used in experiments. However, one should never assume that data from a pig can be assumed to be the same for a human. The movement and deposition of chemicals often differs between human and pig skin.

From my searches, I haven’t been able to find similar research performed on humans. This paper in particular has led to some of the often quoted “rules” about ascorbic acid.

“Ascorbic acid must have a pH below 3.5 for effective penetration.”

Pinnell and his group tested a 15% ascorbic acid solution adjusted to different pHs ranging from 2 to 5. The 15% ascorbic acid solutions also contained 2% zinc sulfate, 0.5% bioflavonoids, 1% hyaluronic acid, and 0.1% citrate.

While the control situation wasn’t described it’s likely either the vehicle (product without the ascorbic acid) or a water solution was applied to the skin. The control measurement shows that there is some inherent levels of ascorbic acid already present in the skin from the diet.

The test solutions were applied to the pig skin using a Hill Top Chamber. A Hill Top Chamber is a small and round disk which is placed on the surface of the skin, the product is placed in the chamber or a piece of fabric is soaked in the testing material, and the entire chamber is then sealed. This reduces loss of product from evaporation and is a common method of performing occlusive test patches.

The ascorbic acid solutions at pH 2.5, 3.0, 3.5, 4.0, and 5.0 were performed on three pigs, however the control, pH 2, and 4.5 were only performed on two pigs.

The Hill Top Chamber was soaked with 0.2 mL of the ascorbic acid solution then sealed for 24 hours. After this period of occlusion, the skin washed then stripped of the stratum corneum and then small pieces of the skin was removed and tested for ascorbic acid content.

As you can see from the data, the amount of ascorbic acid found in the skin was much higher in ascorbic acid solutions at pH 3.5, 3.0, 2.5, and 2.0. The researchers hypothesize that it is due to the pKa of ascorbic acid which is 4.2. When the pH of a solution containing ascorbic acid is lower than its pKa more of the ascorbic acid will be protonated. Protonated ascorbic acid is neutrally charged which may allow it to enter the skin more easily.

It’s important to notice the error bars on the amount of ascorbic acid absorbed at pH 2.0. There is considerable deviation from the mean in the results even though it was only tested on 2 subjects. More test subjects would provide a clearer idea of how much ascorbic acid would penetrate at pH 2 on an average population of pigs.

Statistical differences also weren’t calculated between the data points, for example it’s difficult to tell from the way that the data is presented if there is a change in ascorbic acid content between the control, pH 4.0, 4.5, and 5.0 – even if they look different on the graph. Likewise, it’s difficult to tell if there is an increase in ascorbic acid penetration between pH 3.0 and pH 2.5 – despite the trend with pH 2.0 pushing towards that inference. It’s likely that there is a statistically significant difference between absorption between pH 3.5 and 3.0, but a larger study would provide us with more confident answers.

So based on this data, many further studies and brands have assumed that a pH below 3.5 results in considerable more skin penetration of ascorbic acid on humans – despite these results being performed on pigs, and relative low strength of the study. If the reason why ascorbic acid is more easily absorbed into the skin is due to the pKa then this would likely hold true for humans as well – as pH drops below 4.2, more ascorbic acid becomes protonated, and penetration increases.

This assumption is often presented as fact, which is misleading. It also doesn’t take into account other factors present in a cosmetic product, such as penetration enhancers. Encapsulation, surfactants, and solvents could increase (or decrease) the amount of ascorbic acid absorbed into the skin regardless of the product’s pH.

In this experiment, the stratum corneum was removed before measurements of ascorbic acid to test for deep penetration of ascorbic acid. It’s possible that some of the benefits conferred by topical application of ascorbic acid aren’t facilitated by deep penetration, the antioxidant and photoprotective effect of ascorbic acid may still occur when it is present in or on the stratum corneum. Other benefits like reduction of hyperpigmentation and an increase in collagen production are likely dependent on penetration past the stratum corneum.

Unfortunately I haven’t been able to find further studies on humans or otherwise to provide answers to these questions.

“Ascorbic acid serums must be at least 10% to be effective”

After the first experiment of testing 15% ascorbic acid with different pHs, Pinnell and his group tested how concentration of ascorbic acid affects skin penetration. This time they tested 7 ascorbic acid solutions with varying concentrations all at pH 3.2. The concentrations of the rest of the formulation are assumed to be the same as the previous experiment.

The ascorbic acid solutions were applied in the same manner, with a Hill Top Chamber for 24 hours, followed by washing, stripping, and then assessment.

The maximum amount of ascorbic acid penetration was seen when 20% ascorbic acid at pH 3.2 was used.

All concentrations were tested on 3 pigs, and there is quite a bit of deviation from mean between absorption among the 3 pigs tested. This makes it difficult to assess the true difference in absorption between a 10% and 15% ascorbic acid, and a 15% and 20% ascorbic acid.

Absorption also seemed to peak at 20%, the 25% ascorbic acid solution penetrated less than the 20%, and the 30% even less so. The researchers did not explore or hypothesize on why this occured, and I’ve been unable to find an answer in any later research as well.

While 20% ascorbic acid certainly led to the greatest increase in levels of ascorbic acid, the 5% solution still increased ascorbic acid levels in the pig skin by about 6 fold.

It’s very important to remember that the way that this experiment was performed does not mimic the way that ascorbic acid solutions are often applied to the skin. With the Hill Top Chamber, the solvent’s (in this case water) evaporation is reduced – whereas when we apply it to the skin the solvent evaporates. What this means is that the kinetics of ascorbic acid penetration into the skin may not be the same.

For example, if half of the solvent of a 10% ascorbic acid solution evaporates, it is equivalent to a 20% ascorbic acid solution – the total amount of ascorbic acid by mass is the same, but the concentration has changed. This may mean that we could see a different maximum absorption by concentration in an experiment where the solvent was allowed to evaporate the way that it is often applied.

Human clinical trials with “low” ascorbic acid concentrations, 3% ascorbic acid cream and a 5% ascorbic acid cream, were able to show statistically significant improvements on measurements of photodamage and photoageing in their study groups.

Another thing many people hold on to is the concept that their products must be working at “maximum efficiency”, unfortunately this is unrealistic and there’s going to be variations in the amount of ascorbic acid that penetrates your skin with each application – even the amount that you apply to your skin will vary each time. This is why good cosmetic studies are performed over a longer period of time.

For example, if we look at the 20% concentration, the pig skin concentration of ascorbic acid increased to about 1100 pmol of ascorbic acid per mg of pig skin, which is about 0.19 μg ascorbic acid per mg of pig skin. 1.0 mg of a 20% ascorbic acid (w/w) contains about 1135589.37 pmol of ascorbic acid, if that helps give you a sense of the “efficiency”. In these experiments, 200 μL or 0.2 mL solution was used in total for each application, which contains about 227117874.1767 pmol of ascorbic acid if we assume density of the solution (w/w) is 1.

Higher concentrations of ascorbic acid may lead to more irritation (measured by skin redness or erythema), but I haven’t found any studies that looked at this specifically.

Continued in Skin penetration of Ascorbic Acid: Part II

Source: Pinnell, S. R., Yang, H. , Omar, M. , Riviere, N. M., DeBuys, H. V., Walker, L. C., Wang, Y. and Levine, M. (2001), Topical L‐Ascorbic Acid: Percutaneous Absorption Studies. Dermatologic Surgery, 27: 137-142. DOI: 10.1046/j.1524-4725.2001.00264.x

Anhydrous Vitamin C (Ascorbic Acid) Serum

This is an anhydrous ascorbic acid serum that I’ve been working on!


Ascorbic acid is notoriously unstable in the presence of water, it quickly oxidizes into dehydroascorbic acid which is yellow/orange in colour and (according to current research) isn’t as effective as ascorbic acid in producing skin benefits like evening of skin tone, reducing free radicals and reactive oxygen species, and increasing collagen production.

There are many compounds made from ascorbic acid that are designed to keep the antioxidant more stable. However many of these compounds haven’t been shown to act the same way as ascorbic acid or penetrate the skin. As well, enzymes in the skin are required to convert these compounds back into ascorbic acid, and there is little evidence to show that this occurs to a large extent.

By removing water from the formula, ascorbic acid can be stabilized against oxidation and experiments have shown that it can be so stable that it can resist months of exposure to oxygen (the test formulas were bubbled through with oxygen, like a fish tank!).

By removing water, we often create heavier, stickier, and much shinier products. This prototype, while heavy feeling on the finger, applied with a relatively matte and light finish. Hopefully it will stand up to stability testing, but I was so excited that I had to try it on myself immediately!



I’m excited to tweak this into a more elegant and light formula, especially with dramatic results like these!


From Stability, transdermal penetration, and cutaneous effects of ascorbic acid and its derivatives

So far, so good with the prototype – it’s been almost 2 weeks and yet to change colour. The smaller bottle contains unstabilized ascorbic acid in water.



Can you use Niacinamide and Vitamin C (Ascorbic Acid) together?

Yes! (For most people.)

Niacinamide and ascorbic acid on their own are great skin care ingredients. Ascorbic acid is not only a good water-soluble antioxidant, but may also increase production of collagen in the skin which can help mitigate some of the damaging effects caused by UV exposure.

Niacinamide is a good anti-inflammatory which makes it useful for treating inflammatory conditions like acne and has been shown to increase naturally occurring moisturizers in the skin.

They’re both very effective at reducing hyperpigmentation of the skin – it’s easy to see why people would want to use the two of them together.


If you Google Niacinamide and Vitamin C, you’ll get some posts about not combining them. The worry is that they form a 1:1 complex and cancel out each other’s skin benefits.

There are two main chemical reactions that occur when you mix niacinamide and ascorbic acid. The first occurs when you mix niacinamide with any acid (or base), and that’s the conversion of niacinamide to niacin. This takes a long time and is more of a concern for manufacturers that are using niacinamide in acidic products.

It’s possible that niacin is just as effective as niacinamide, but it has the side effect of activating Langerhan cells in the skin. This leads to the release of prostaglandins and dilation of the blood vessels, which causes the skin to flush with redness and tingle. This flushing and tingling is temporary, but can be uncomfortable and potentially problematic for someone with inflammatory acne or erythema.

The second concern is the formation of a complex between niacinamide and ascorbic acid. I think the worry is that this complex is no longer effective in benefiting the skin, and I’ve also seen concerns that it may produce hydrogen peroxide which can lead to skin cell death.

I took a look at the research to get a better understanding of these two chemical reactions. It is important to note that a few of these papers are old, so they don’t use newer visualization techniques that can help us quantify chemical changes and structures more accurately.

Hydrolysis of Niacinamide to Niacin

Amides, like niacinamide, are very stable compounds. This stability means it takes a lot of heat and concentrated acids or bases to get niacinamide to turn into niacin (also known as nicotinic acid).

I’m going to do a separate post that will explain this more, but the important thing to see is that the -NH2 group, a nitrogen and two hydrogens, is replaced with an -OH group, an oxygen and a hydrogen.

In fact, niacinamide is so stable, that a 10% solution can be heated to 120 °C (248 °F) for 20 minutes with no niacin forming.

In another experiment it took about 75 hours in a pH 2 solution at 90 °C (194 °F)  to convert about half the niacinamide to niacin.

Heat vastly speeds up reactions; a rough approximation is that every 10 °C (18 °F)   increase in temperature doubles the reaction rate. If the pH 2 solution was kept at 30 °C (86 °F)   we could expect it to take over 4800 hours or almost half a year – and that’s if the acidic solution didn’t corrode your skin first!

An experiment with temperature conditions closer to room temperature shows how heat affects the rate of conversion. Only after 6 weeks of storage at 45  °C (113 °F) in acidic pHs did they find niacin, and the conversion never exceeded 2%. At room temperature (25 °C or 77 °F) the amount of niacin was less than 1% after 6 weeks, but the measurement wasn’t accurate enough to tell exactly how much, just that a small amount was present.

Another thing to keep in mind is that reaction rates also slow down when the viscosity or thickness of the solution is increased. Most skin care products, even serums, are thickened to some extent – which will further slow down the conversion of niacinamide to niacin.

If we can get metaphorical for a second, chemical reactions aren’t like a distance race. They’re more like jumping over hurdles. There’s a minimum amount of energy that’s needed to cause the molecule to change, called the activation energy. If you don’t have the energy to jump over a hurdle, you can’t progress. You can’t just roll on the ground until you hit the finish line.

What all this means is that products formulated with niacinamide and an acid may produce some niacin over time. This can be a problem for some, as even a miniscule amount of niacin can produce some faint skin redness, as little as 0.001% – amounts in the range of 0.01% to 0.1% will produce much more apparent skin redness for more people.

It’s also important to note the difference between irritation due to an acidic product versus the flushing caused by niacin. Niacin flushing appears evenly, affecting everywhere it’s been applied. It is also temporary (often lasting longer than 20 minutes), and comes with a tingling sensation.

If you’re using a product with niacinamide and ascorbic acid in the ingredients list and aren’t noticing any skin reddening…there’s either very little niacin that’s been formed, or you’re not very sensitive. If you’re worried about a product that contains both ingredients, storing it in a cool environment will discourage transformation of niacinamide to niacin.

But what about applying a product with niacinamide and then one with ascorbic acid? Is niacin being formed? I don’t want to say absolutely not, but the amount potentially formed is so miniscule, I don’t think it’s a relevant worry.

Niacinamide and Vitamin C (Ascorbic Acid) Complex

Mixing niacinamide and ascorbic acid turns the two clear solutions a yellow colour, but this isn’t the same yellow colour that appears when ascorbic acid is oxidized into dehydroascorbic acid.

This yellow color is due to the formation of a charge-transfer complex, called niacinamide ascorbate. An electron is transferred from the ascorbic acid to the niacinamide – this weakly holds the two together.

I think the fear of this complex negating the benefits of both ascorbic acid and niacinamide is not knowing that this complex is easily reversed and separated.

An experiment found the association equilibrium, or the balance of free ascorbic acid and niacinamide to niacinamide ascorbate, to be 1.45 at 25 °C (77 °F) .

This experiment also found that the pH of the solution that the two are mixed together in affects how much of the niacinamide ascorbate is formed. Niacinamide ascorbate forms best at a pH close to 3.8, but at pHs higher or lower less is formed. This means if the pH of a solution they were mixed together in was changed, away from 3.8, the two molecules would separate from each other.

This means if we added the same number of molecules of niacinamide and ascorbic acid into a solution of pH 3.8, about half of them would be in the niacinamide ascorbate complex, and the rest of them would just be on their own floating around.

I’m going to do a separate post that will walk through the calculation of this.

The pH dependent formation is important, because only the surface of our skin is acidic. Each deeper layer of skin becomes less and less acidic, finally reaching physiological pH which is around 7. This means as the niacinamide ascorbate moves deeper into the skin, there’s less impetus for them to be associated with each other. As well, as more individual ascorbic acid and niacinamide molecules move into the skin, the ratio of free to complexed niacinamide and ascorbic acid would decrease, and the complexed niacinamide ascorbate would separate until equilibrium was reached again.

One study that used niacinamide ascorbate on human skin explants found that it was still able to reduce melanin production in response to UV exposure. So it would seem its active effects aren’t hampered by being complexed together.

Another experiment found that the niacinamide ascorbate complex helped stabilize the ascorbic acid against degradation.

So the important takeaway is to know that the yellow colour isn’t the same thing as an oxidation of ascorbic acid, and that the complex formed between niacinamide and ascorbic acid is completely reversible. The niacinamide and ascorbic acid are held together like two pieces of paper with static, versus glue.

There is one paper that is cited to say that UV exposure caused the production of hydrogen peroxide in the skin when niacinamide and ascorbic acid were used together, however the effect was small and the proposed peroxyl radical mechanism is theoretical.

Many skin care ingredients will degrade or change with UV exposure, but using a sunscreen will greatly reduce or even eliminate the chances of this occurring.

The paper also theorized that by mixing the niacinamide and ascorbic acid with tocopherol (Vitamin E), the radical production could be mitigated.


I hope this has helped you understand what’s occurring when you mix niacinamide and ascorbic acid (or any acid) together. While there are chemical reactions that occur, they either occur super slowly, or are reversible.

You can definitely still choose to use niacinamide and ascorbic acid separately. You could use ascorbic acid during the day and niacinamide in the evenings. If you do choose to use them together, I don’t think the evidence points to any disabling of their benefits or any skin damage.

People do often experience some redness or tingling when using the two, but I think that can be chalked up to the irritation caused by ascorbic acid, especially if the product isn’t pH adjusted and acidic. I don’t want to suggest you to take a niacin pill to experience a flush, but unless the redness is very even and demarcated, it’s not likely due
to niacin.

Thank you to Jonathon Moir and James LaFortune for their expertise and help in writing this article